Gadolinium complex comprising DO3A-tranexamic acid conjugate

Abstract

The present disclosure relates to a magnetic resonance imaging (MRI) contrast agent containing a gadolinium complex, and more specically, to a DO3A-tranexamic acid compound having a structure of a chemical formula 1, or an ester compound thereof, and gadolinium complexes thereof. The DO3A-tranexamic acid compound or the ester compound thereof may be used to prepare gadolinium complexes. The gadolinium complexes exhibit thermodynamic and kinetic stabilities, and show the relaxation rate equal to that of the clinical contrast agent which is currently commercially available. Therefore, the gadolinium complexes can be widely used as an MRI contrast agent.

Claims

1. A DO3A-tranexamic acid compound having a structure of a following chemical formula 1, or an ester compound thereof: ##STR00004## where R is H, NH.sub.2 or CONN (CH.sub.2).sub.2NH.sub.2.

2. A method for producing the DO3A-tranexamic acid or the ester compound thereof of claim 1, wherein RCONH(CH.sub.2).sub.2NH.sub.2 (compound 1c), wherein the method comprises: a) adding and agitating bromoacetyl bromide into trans-4 (aminomethyl)cyclohexaneethylcarboxylate hydrochloride to form a first mixture; b) adding and agitating DO3A-(.sup.tBuO).sub.3 to the first mixture to produce DO3A (.sup.tBuO).sub.3 tranexamic ethyl ester conjugate; c) adding 1,2-diaminoethane to the DO3A (.sup.tBuO).sub.3 tranexamic ethyl ester conjugate to form a second mixture; d) removing a solvent from the second mixture under a low pressure and then dissolving the resultant product in methanol and then performing silica gel chromatography thereto; e) adding TFA to the product subjected to the chromatography to deprotect a tert-butyl group; and f) drying the thus-resulting product in a vacuum state to obtain the DO3A-tranexamic acid or the ester compound thereof.

3. A method for producing the DO3A-tranexamic acid or the ester compound thereof of claim 1, wherein RNH.sub.2 (compound 1d), wherein the method comprises: a) adding and agitating di-tert-butyl dicarbonate to trans-1,4-diaminocyclohexane to form a first mixture: b) adding and agitating bromoacetyl bromide to the first mixture to form a second mixture; c) adding and agitating DO3A-(.sup.tBuO).sub.3 to the second mixture to produce DO3A (.sup.tBuO).sub.3 tranexamic amine conjugate; d) removing a solvent from the resultant product under a low pressure and then dissolving the resultant product in methanol and then performing silica gel chromatography thereto; e) adding TFA to the product subjected to the chromatography to deprotect a tert-butyl group; and f) drying the thus-resulting product in a vacuum state to obtain a DO3A-tranexamicamine compound.

4. A method for producing the DO3A-tranexamic acid or the ester compound thereof of claim 1, wherein RH (compound 1e), wherein the method comprises: a) adding and agitating bromoacetyl bromide to (aminomethyl)cyclohexane to prepare a first mixture; b) adding and agitating DO3A-(.sup.tBuO).sub.3 to the first mixture to prepare DO3A-(.sup.tBuO).sub.3 tranexamic conjugate; c) removing a solvent from the resultant product under a low pressure and then dissolving the resultant product in methanol and then performing silica gel chromatography thereto; d) adding TFA to the product subjected to the chromatography to deprotect a tert-butyl group; and e) drying the thus-resulting product in a vacuum state to obtain the DO3A-tranexamic acid or the ester compound thereof.

5. A composition for a complex ligand (L), wherein the composition contains the DO3A-tranexamic acid or the ester compound thereof of claim 1.

6. A complex containing the DO3A-tranexamic acid or the ester compound thereof of claim 1 as a ligand (L), wherein the complex contains a metal atom coordinated with the ligand.

7. The complex of claim 6, where the metal atom is gadolinium (Gd).

8. A MRI contrast agent containing the complex of claim 6 as an effective component.

9. The MRI contrast agent of claim 8, wherein the contrast agent has ECF (extracelluar fluid) image contrast function.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings, which are incorporated in and form a part of this specification and in which like numerals depict like elements, illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure.

(2) FIG. 1 shows a synthesis diagram of a DO3A-tranexamic acid (1c) and a gadolinium complex (2c) thereof in accordance with the present disclosure.

(3) FIG. 2 shows a synthesis diagram of a DO3A-tranexamic acid (1d) and a gadolinium complex (2d) thereof in accordance with the present disclosure.

(4) FIG. 3 shows a synthesis diagram of a DO3A-tranexamic acid (1e) and a gadolinium complex (2e) thereof in accordance with the present disclosure.

(5) FIG. 4 shows a graph illustrating an evolution of R.sub.1P (t)/R.sub.1P (0) for various MRI contrast agents as a function of a time.

(6) FIG. 5A shows coronal and axial T1-weighted images of ICR mice in five minutes after the present contrast agent is injected thereto, where K indicates a kidney; A indicates abdominal aorta.

(7) FIG. 5B shows coronal and axial T1-weighted images of ICR mice in one hour after the present contrast agent is injected thereto, where A indicates heart; L=liver; B=urinary bladder; G=gallbladder.

(8) FIG. 6 shows a relative cell toxicity (%) of a normal conjunctival fibroblast obtained using Gd-DOTA and 2b-2. A standard deviation (SD) is obtained using a triple analysis (n=3).

(9) FIG. 7A to FIG. 7E show ITC determination of binding thermodynamics of Gd.sup.3+ to the ligand. A binding isotherm corresponds to a plot of an integral heat as a function of a molar ratio of Gd.sup.3+/1a-e. A solid line corresponds to the best fit curve of data into one set of mathematically determined site models and indicates one molecule (Gd.sup.3+) being coupled to a respective ligand. A dissociation constant (Kd), an association constant (Ka), the number of Gd.sup.3+ coupled to a single ligand (N), and a united enthalpy change (H) are obtained by analysis of data. The dissociation and association constants are critical factors for determining a relative stability compared to a ligand (1a-e).

(10) FIG. 8 shows CNR profiles for a liver and kidney, obtained using various contrast agents.

(11) FIG. 9 shows an evolution of R.sub.1 for 2 ([Gd]=1.0 mM) in PBS as a function of an incubation time under different pH conditions.

DETAILED DESCRIPTIONS

(12) Examples of various embodiments are illustrated in the accompanying drawings and described further below. It will be understood that the description herein is not intended to limit the claims to the specific embodiments described. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the present disclosure as defined by the appended claims.

(13) In present embodiments, all reactions were performed under a dinitrogen atmosphere using a standard Schlenk techniques. Solvents were purified and dried by standard procedures. 1,4,7,10-tetraazacyclododecane (DOTA) was purchased from Strem (US), and trans-4-(aminomethyl)-cyclohexane carboxylic acid (tranexamic acid) and trans-1,4-diamino cyclohexane were purchased from Aldrich, and aminomethyl cyclohexane was purchased from TCI. All other commercial reagents were purchased and received and used from Aldrich unless otherwise specified. deionized water (DI water) was used for all experiments. A 1H experiment was carried out on a Bruker Advance 400 or 500 spectrometer in KBSI. Chemical shifts were given as a value as a comparison value to tetramethylsilane (TMS) as an internal standard. A coupling constant is represented by Hz, and FAB mass spectra were obtained using a JMS-700 model (Jeol, Japan) mass spectrophotometer. MALDI-TOF mass spectra were obtained using a Voyager DE-STR (Applied Biosystems, U.S.). Elemental analysis was performed at the Center for Scientific Instruments of KNU (Kyungpook National University).

(14) ##STR00003##

(15) Embodiment 1: Ligand Synthesis

(16) 1) Synthesis of Compounds 1a and 1b

(17) The compounds 1a and 1b are prepared using a previous document (Gu, S.; Kim, H. K.; Lee, G. H.; Kang, B. S.; Chang, Y.; Kim, T. J. J. Med. Chem. 2011, 54, 143).

(18) 2) Synthesis of Compound 1c

(19) First, using a previous document method (Gu, S.; Kim, H. K.; Lee, G. H.; Kang, B. S.; Chang, Y.; Kim, T. J. J. Med. Chem. 2011, 54, 143), DO3A (.sup.tBuO).sub.3 tranexamic ethyl ester conjugate (6.8 mmol) (cf, RCO.sub.2Et, Chart 1) is prepared. The DO3A (.sup.tBuO).sub.3 tranexamic ethyl ester conjugate is added to a neat 1,2-diaminoethane (2.3 mL, 33.8 mmol) to form a mixture. The mixture is agitated at a room temperature (RT) for 72 hours, to which chloroform is added, and, the resultant product is extracted using water (30 mL3 times). The organic extract is dried on MgSO.sub.4, and filtered and evaporated to produce a crude product which in turn, is subjected to a silica chromatography (gradient elution CH.sub.2Cl.sub.2 to 10% MeOHCH.sub.2Cl.sub.2, R.sub.f=0.6 (MeOH/CH.sub.2Cl.sub.2=2:8)) to obtain a yellow solid. This solid is dissolved in (10 mL) and, then, trifluoroacetic acid (TFA) was added to the solution to deprotect a tert-butyl group. The mixture is agitated for one night at a room temperature, and, then, the solvent is removed from the mixture solution in a vacuum state to leave only an oily residue. The, water is absorbed into this residue. Then, acetone is added thereto to allow a white solid to precipitate as a product. Then, the product is subjected to multiple acetone cleanings, and, then, dried at a vacuum state: yield: 0.36 g (92%).

(20) 1H NMR (D.sub.2O): =3.82 (s, 4H, NCH.sub.2CO.sub.2), 3.62 (2H, NCH.sub.2CO.sub.2), 3.67 (s, 2H, CH.sub.2CON), 3.44 (m, 10H, overlapped NCH.sub.2CH.sub.2N in the ring (8H) & CONHCH.sub.2 (2H)), 3.08 (d, 2H, CONHCH-2C), 2.24/1.52 (m, 2H, CONH), 1.85/1.41 (m, 8H, CH.sub.2, cyclohexyl), 1.03 (m, 2H, CH.sub.2, cyclohexyl). Anal. Calcd for C.sub.26H.sub.47N.sub.7O.sub.82H.sub.2O: C, 50.23; H, 8.27; N, 15.77. Found: C, 50.66; H, 8.24; N, 16.01. MALDI-TOF MS (m/z): Calcd for C.sub.26H.sub.47N.sub.7O.sub.8: 585.35, Found: 586.74 ([MH]+), 608.70 ([MNa]+).

(21) 3) Synthesis of Compound 1d

(22) First, DO3A- (.sup.tBuO).sub.3 (1.6 g, 3.1 mmol) is added to trans-[4-(2-chloroacetylamino)cyclohexyl]carbamic tert-butyl ester (1.0 g, 3.4 mmol) solution among chloroform (100 mL) prepared from trans-1,4-cyclohexane using a previous document method (Lee, D. W.; Ha, H. J. Synthetic Commun 2007, 37, 737 Roth, G. J.; Heckel, A.; Colbatzky, F.; Handschuh, S.; Kley, J.; Lehmann-Lintz, T.; Lotz, R.; Tontsch-Grunt, U.; Walter, R.; Hilberg, F. J. Med. Chem. 2009, 52, 4466). The mixture is agitated at a room temperature for 24 h, and all solids are removed and a filtered liquid is evaporated in a vacuum to leave a yellow oily residue. This residue is subjected to silica column chromatography (gradient elution: CH.sub.2Cl.sub.2 to 10% MeOHCH.sub.2Cl.sub.2, R.sub.f=0.4 (MeOH/CH.sub.2Cl.sub.2=1:9)) to get an off-white solid. This solid is re-dissolved in dichloromethane and then a considerable amount of TFA is added thereto. After the product is agitated for one night, and a methanol is added thereto to precipitate a white solid. Then, the final product is more purified using a typical purification method: yield: 0.62 g (87%).

(23) 1H NMR (D.sub.2O): =3.72 (m, 10H, overlapped NCH.sub.2CO.sub.2 (8H), CONHCH & H.sub.2NCH (2H)), 3.40/3.21 (m, 16H, NCH.sub.2CH.sub.2N), 2.06/1/55/1.38 (m, 8H, CH.sub.2, cyclohexyl). Anal. Calcd for C.sub.22H.sub.40N.sub.6O.sub.72CF.sub.3COON2H.sub.2O: C, 39.00; H, 6.29; N, 10.50. Found C, 39.13; H, 6.05; N, 9.35. MALDI-TOF MS (m/z): Calcd for C.sub.22H.sub.40N.sub.6O.sub.7: 500.30, Found: 501.33 [MH]+, 523.33[MNa]+.

(24) 4) Synthesis of Compound 1e

(25) First, DO3A-(.sup.tBuO).sub.3 (3.0 g, 5.8 mmol) is added to 2-cloro-N-cyclohexilmethylacetamid (1.2 g, 6.4 mmol) solution among acetonitrile (30 mL) prepared using a previous document method (Cho, S. D.; Song, S. Y.; Kim, K. H.; Zhao, B. X.; Ahn, C.; Joo, W. H.; Yoon, Y. J.; Falck, J. R.; Shin, D. S. B Kor. Chem. S C. 2004, 25, 415). The mixture is agitated at a room temperature for 24 h, and all impurity solids are removed and a filtered liquid is evaporated in a vacuum to leave a yellow oily residue. This residue is subjected to a silica column chromatography (gradient elution: CH.sub.2Cl.sub.2 to 10% MeOHCH.sub.2Cl.sub.2, R.sub.f=0.4 (MeOH/CH.sub.2Cl.sub.2=1:9)). Then, the evaporation is applied thereto under a pressure reduction to obtain an off-white solid. As in the synthesis of the compound 1d, the TFA is added thereto to deprotect a tert-butyl group to obtain an off-white solid as a final product. yield: 2.4 g (82%).

(26) 1H NMR (D.sub.2O): =3.74/3.57 (m, 8H, NCH.sub.2CO.sub.2), 3.30 (m, 10H, overlapped NCH.sub.2CH.sub.2N (8H) & CONHCH.sub.2 (2H)), 3.10 (m, 8H, NCH.sub.2CH.sub.2N), 1.98/1.44/1.27 (m, 4H, CH.sub.2, cyclohexyl), 1.88 (m, 1H, NHCH.sub.2CH). Anal. Calcd for C.sub.22H.sub.39N.sub.5O.sub.73CF.sub.3COOH3H.sub.2O: C, 38.14; H, 5.49; N, 7.94. Found: C, 37.83; H, 5.76; N, 8.44. MALDI-TOF MS (m/z): Calcd for C.sub.22H.sub.39N.sub.5O.sub.7,: 485.28, Found: 486.42 ([MH]+), 508.44 ([MNa]+).

(27) Embodiment 2: Synthesis of Gd Complex

(28) 1) Synthesis of Complexes 2a and 2b

(29) The complexes 2a and 2b are prepared using a previous document (Gu, S.; Kim, H. K.; Lee, G. H.; Kang, B. S.; Chang, Y.; Kim, T. J. J. Med. Chem. 2011, 54, 143).

(30) 2) Synthesis of Complex 2c

(31) First, gadolinium chloride (0.64 g, 1.7 mmol) is added to water (50 mL) 1c (1.0 g, 1.7 mmol) solution and the mixture is agitated at 50 C. for 48 h. pH is checked periodically and thus the pH is adjusted to 7.0 to 7.5 using 1 N NaOH. The reaction mixture is dried at a vacuum state to leave an oily residue. Water is absorbed in the residue. Acetone is added thereto to precipitate a white solid. This solid is filtered and cleaned using acetone and the product is dried at a vacuum state. A hygroscopic ivory solid is obtained as a final product: yield: 0.75 g (86%).

(32) Anal. Calcd for C.sub.26H.sub.44GdN.sub.7O.sub.82CF.sub.3COOH8H.sub.2O: C, 32.40; H, 5.62; N, 8.82. Found: C, 31.99; H, 5.22; N, 9.00. HR-FABMS (m/z): Calcd for C.sub.26H.sub.45GdN.sub.7O.sub.8, 741.26 ([MH H.sub.2O]+). Found: 741.2567; Calcd for C.sub.26H.sub.44GdN.sub.7O.sub.8Na, 763.24 ([MNa H2O]+). Found: 763.2397.

(33) 3) Synthesis of Complex 2d

(34) This synthesis process is the same as in the synthesis process in the complex 2c except that the compound 1c is replaced with the compound 1d. A hygroscopic off-white solid is obtained as a final product: yield: 1.15 g (88%).

(35) Anal. Calcd for C.sub.22H.sub.37GdN.sub.6O.sub.72CF.sub.3COOH5H.sub.2O: C, 32.10; H, 5.08; N, 8.64. Found: C, 31.55; H, 5.09; N, 9.06. HR-FABMS (m/z): Calcd for C.sub.22H.sub.38GdN.sub.6O.sub.7, 656.21 ([MH H2O]+). Found: 656.2045; Calcd for C.sub.26H.sub.44GdN.sub.7O.sub.8Na, 678.19 ([MNa H2O]+). Found: 678.1867.

(36) 3) Synthesis of Complex 2e

(37) This synthesis process is the same as in the synthesis process in the complex 2c except that the compound 1c is replaced with the compound 1e. A hygroscopic off-white solid is obtained as a final product: yield: 0.75 g (86%).

(38) Anal. Calcd for C.sub.23H.sub.38GdN.sub.5O.sub.74H.sub.2O: C, 38.06; H, 6.39; N, 9.65. Found: C, 38.59; H, 6.09; N, 9.69. HR-FAB MS (m/z): Calcd for C.sub.23H.sub.39GdN.sub.5O.sub.7, 655.21 ([MH H.sub.2O]+). Found: 655.2093.

(39) Embodiment 3: Relaxation Rate Measurement

(40) T1 measurement is carried out using an inversion recovery method at 1.5 T (64 MHz) in a variable inverse time (TI). MR images are obtained for 35 different TI values in a range of 50 to 1750 ms. A T1 relaxation time is obtained from a nonlinear least-squares fit of a signal strength measured at each TI value. For a T2 CPMG (Carr-Purcell-Meiboon-Gill) pulse sequence measurement, the measurement is adapted to a multi spin-echo measurement. 34 images are obtained at 34 different echo time (TE) values in a range of 10 to 1900 ms. The T2 relaxation time is obtained from a nonlinear least-squares fit of mean pixel values for multi spin-echo measurement at each echo time. Next, a relaxation rate (R.sub.1 and R.sub.2) is calculated as an inverse of a relaxation time per mM. The determined relaxation time (T1 and T2) and relaxation rate (R.sub.1 and R.sub.2) are subjected to an image processing to get a relaxation time map and relaxation rate respectively.

(41) The complexes 2a-e exhibit R1 relaxation rates with PBS which are comparable to those of clinically available MRI contrast agents such as Gd-DOTA (Dotarem) and Gd-BT-DO3A (Gadovist) or which are superior to those of the Gd-DOTA (Dotarem) and Gd-BT-DO3A (Gadovist) (Tablet). For comparison between the complexes 2a-e, there is no noticeable difference in R.sub.1's. The measurement is carried out for two different media, that is, PBS (pH=7.4) and PBS solution of HAS in order to indicate which blood-pool effect is expressed by the complexes 2a-e. Compared to R.sub.1's in the PBS, R.sub.1 in the HAS exhibits meaningful increase, which indicates that there is a little interaction between the present new series and the HAS.

(42) TABLE-US-00001 TABLE 1 in PBS and 0.67 mM HAS, relaxation rate data of complexes 2a-e, Gd-DOTA and Gd-BT-DO3A (64 MHz, 293K) R.sub.1 (mM.sup.1s.sup.1) R.sub.2 (mM.sup.1s.sup.1) R.sub.1 R.sub.2 in 0.67 in 0.67 (mM.sup.1s.sup.1) (mM.sup.1s.sup.1) mM HSA mM HSA 2a 4.84 0.18 4.91 0.25 5.11 0.10 6.04 0.13 2b 3.87 0.11 3.98 0.26 3.98 0.14 5.43 0.28 2c 3.68 0.13 3.62 0.26 3.74 0.07 4.10 0.07 2d 3.94 0.13 3.68 0.29 3.91 0.09 4.07 0.09 2e 4.49 0.18 4.52 0.28 5.21 0.23 6.22 0.28 Dotarem* 3.59 0.17 3.87 0.20 4.05 0.13 4.30 0.25 Gadovist* 4.38 0.15 4.27 0.30 4.81 0.17 5.54 0.24

(43) Embodiment 4: Transmetalation Kinetics

(44) This experiment is prepared using a previous document method (Laurent, S. Elst, L. V.; Copoix, F.; Muller, R. N. Investigative radiology 2001, 36, 115). This is based on measurements of evolution of a water proton longitudinal relaxation rate (R.sub.1P) of a buffer solution (phosphate buffer, pH 7.4) containing 2.5 mmol/L gadolinium complex and 2.5 mmol/L ZnCl.sub.2. Next, 10 L ZnCl.sub.2 250 mmol/L solution is added to a 1 nk paramagnetic complex buffer solution. The mixture is strongly agitated, and then, 300 L thereof is taken for a relaxation measurement (relaxometric measurement). Comparison the present series with Gd-DOTA, Gd-BOPTA, Gd-DTPA-BMA, and Gd-EOB-DTPA shows the same results from the presence of ZnCl.sub.2. A R.sub.1P relaxation rate is acquired by excluding a diamagnetic contribution of a water proton relaxation from the measured relaxation rate R.sub.1=(1/T1). The measurement is performed at a room temperature on a 3T whole body system (Magnetom Tim Trio, Simens, Korea Institute of Radiological & Medical Science).

(45) FIG. 4 shows a graph illustrating an evolution of R.sub.1P (t)/R.sub.1P (0) for various MRI contrast agents as a function of a time.

(46) An evaluation of a normal paramagnetic longitudinal relaxation rate R.sub.1P (t)/R.sub.1P (0) is observed between the present series 2a-e and comparison examples Dotarem, Primovist, Multihance, and Omniscan (FIG. 4). The used complex in the test is divided into two following groups based on an evolution pattern: (i) a group employing a macrocyclic chelate (ii) a group employing an acyclic chelate. The present series complexes employ the macrocyclic chelate and may be highly likely to have very high kinetic inertness and, thus, keep a 90% or larger portion of the paramagnetic relaxation rate at an initial measurement for 72 h. However, for the group employing the acyclic chelate, the relaxation rate is remarkably reduced. For example, Primovist, and Multihance has a rapid slope to keep only a 50% portion of the paramagnetic relaxation rate at an initial measurement for 72 h.

(47) Embodiment 5: Isothermal Titration Calorimeter (ITC)

(48) In order to quantify binding isotherms of paramagnetic metal ions Gd.sup.3+ with a ligand solution, an ITC test is executed at a VP-ITC isothermal titration microcalorimeter (Microcal, USA). Data collection, analysis and plotting are executed with help of software package Origin, version 8.0 from Microcal. A sample cell has a 1.43 mL volume. The cell is filled with a water-soluble buffer solution of the above compound 1a-e ligand (0.2 mM). A water-soluble buffer solution of Gd.sup.3+ (1.0 mM) is input into a 300 L continuous agitated (300 rpm) syringe and 10 L aliquots thereof is injected into the cell. At a 3-mins interval, the cell is delivered over 20 s. Data points are collected on a 2 s basis. The measurement is executed at 25 C. All titrations are executed three times to secure data consistency and solution stability. These titration isotherms are integrated to find out an enthalpy change at each injection. In order to fit well with two mathematically different sets of site models, the titration isotherms are analyzed. Via a calorie measurement, parameters including a binding constant (Ka), a change in enthalpy (H), a stoichiometry of binding (N) are calculated. Next, a free energy (G) and an entropy change (S) are calculated using a following complexation equation:
Mn++nL.Math.MLn:G=RT.Math.InK.sub.a=H-TS,

(49) where, R indicates a universal gas constant; T is Kelvin temperature.

(50) It may be noted that due to the fact that the present series have fourth order high binding constants (K.sub.a) from measurements using ITC (Isothermal titration calorimeter), the present series have high thermodynamic stability (See Tablet and FIG. 7). For comparisons between the present series, the compound 1b expresses the highest preference to Gd.sup.3+ ions for an unknown reason.

(51) FIG. 7A to FIG. 7E show ITC determination of binding thermodynamics of Gd.sup.3+ to the ligand. A binding isotherm corresponds to a plot of an integral heat as a function of a molar ratio of Gd.sup.3+/1a-e. A solid line corresponds to the best fit curve of data into one set of mathematically determined site models and indicates one molecule (Gd.sup.3+) being coupled to a respective ligand. A dissociation constant (Kd), an association constant (Ka), the number of Gd.sup.3+ coupled to a single ligand (N), and a united enthalpy change (H) are obtained by analysis of data. The dissociation and association constants are critical factors for determining a relative stability compared to a ligand (1a-e).

(52) TABLE-US-00002 TABLE 2 titration data of Gd.sup.3+ (0.2 mM) into water and water-soluble compounds 1a-e (1.0 mM) at 25 C. and pH = 4.5 1a 1b 1c 1d 1e K.sub.a (10.sup.4M.sup.1) 5.19 25.10 5.97 3.41 8.71 K.sub.d (10.sup.6M) 19.27 3.98 16.75 29.33 11.48 N 0.89 0.37 0.28 0.19 0.49 H (kcal .Math. mol.sup.1) 4.19 7.13 5.94 4.33 3.84 S (cal .Math. mol.sup.1 .Math. K.sup.1) 35.60 48.60 41.80 35.30 35.50 G (kcal .Math. mol.sup.1) 6.42 7.35 6.52 6.19 6.74

(53) Embodiment 6: In Vivo MR Experiment

(54) The in vivo experiment is executed with compliance to a regulation of Animal Research Committee of KNU. 29-31 g weight 6-weeks male ICR mice are used. The mice (n=3) are anesthetized with 1.5% isoflurane in oxygen. The measurement is carried out before and after the injection of the complexes 2a-e through the tail vein. The amount of the contrast agent per each injection is as follows: 0.1 mmol Gd/kg for a MR image. After the mice wake from the anesthesia after each measurement, the mice were placed in a cage capable of free access to feed and food. For this measurement, the animals are held at about 37 C. using a warm water blanket. MR images are taken using a 1.5 T MR unit (GE Healthcare, Milwaukee, Wis.) with a manual RF coil for a small animal. The coil may be of a receiver type having a 50 mm inner diameter. Imaging parameters of the spin echo (SE) are as follows: repetition time (TR)=300 msec; echo time (TE)=13 msec; 8 mm field of view (FOV); 192128 matrix size; 1.2 mm slice thickness; acquisition number (NEX)=8. MR images are collected for 24 h after the injection. Anatomical positions having an improved contrast are set for a liver, a bile duct, and a kidney on (post contrast) MR images. For quantitative measurements, a signal intensity in a region of interest (ROI) is measured using Advantage Window software (GE medical, U.S.). The CNR is calculated using a following equation (1):
CNR=SNR.sub.postSNR.sub.pre (1)

(55) where SNR is a sound-to-noise ratio.

(56) FIG. 5A shows coronal and axial T1-weighted images of ICR mice in five minutes after the present contrast agent is injected thereto, where K indicates a kidney; A indicates abdominal aorta. FIG. 5B shows coronal and axial T1-weighted images of ICR mice in one hour after the present contrast agent is injected thereto, where A indicates heart; L=liver; B=urinary bladder; G=gallbladder.

(57) FIG. 8 shows CNR profiles for a liver and kidney, obtained using various contrast agents.

(58) For the complexes 2a to 2e, a strong signal enhancement is observed at the heart and abdominal aorta within 5 mins (FIG. 5A). Among the complexes 2a to 2e, the complexes 2a to 2c exhibit signal enhancements for initial 1 hour for a liver. Thus, the complexes 2a to 2c may be comparable to the typical liver-specific contrast agents such as Primovist and Multihance. However, compared to Primovist and Multihance, only the complex 2a may be discharged via the bile. Thus, only the complex 2a may be considered as a true liver-specific contrast agent (FIG. 5B). Although the complex 2a also may be discharged via the bile, the signal strength is insufficient to be qualified as the liver-specific contrast agent. The complex 2a has the signal strength as weak as that of Gadovist as a typical ECF contrast agent (FIG. 5A). Although the complex 2d expresses a strong signal enhancement at both the kidney and abdominal aorta initially, the complex 2d is completely discharged within one hour like Gadovist. Thus, the complex 2d may be considered as a ECF contrast agent. Although the complexes 2b and 2c express a strong signal enhancement at the liver (FIG. 5A and FIG. 8), a discharge thereof via the bile is not observed. In order to describe this abnormal behavior of the complexes 2b and 2c, two following assumptions are set: the complexes 2b and 2c are input through organic anion transporting polypeptide (OATP) into the liver cells and then, multi-drug resistance protein 2 (MRP2) present as transporting molecules in a membrane between the cells can's transport the complexes 2b and 2c; or the multi-drug resistance protein 2 (MRP2) together with the complexes 2b and 2c may return through the MRP3 or bidirectional OATP to liver sinusoids.

(59) It is noted that for the Primovist and Multihance as typical liver-specific contrast agent, a passive diffusion takes place through OATP1 present in the base membrane in normal liver cells. Then, the agent may be discharged to the bile via MRP2 action. For the Primovist and Multihance, the presence of a lipophilic ethoxy benzyl is believed to play an important role in the above process. In this respect, it is possible that, for the complex 2a, the presence of lipophilic cyclohexil may play a similar role to the above. This issue requires an additional research.

(60) Embodiment 7: Cell Viability Measurement

(61) Normal conjunctival fibroblast cells are used. The cells are kept in DMEM (Gibco) supplemented with heat-inactivated FCS (10%), penicillin (100 IU/mL), streptomycin (100 mg/mL), and gentamicin (200 mg/mL) (all of which are purchased from Gibco). The medium is replaced every 2 days, and the cells are dispensed in a 96-well plate (1104 cells/well/200 L). The contrast agent with various Gd (III) concentrations (50 to 500 M) is added into a serum-free culturing medium and is incubated for 24 h. Next, CCK-8 (10 L) is added to each well and then, the cell viability is evaluated. The solution is removed at 37 C. after 4 h. Using a microplate reader (Molecular Device, USA Bio-rad Reader 550), an O.D. (Optical Density) value is read at 450 nm and the cell viability/toxicity is determined.

(62) FIG. 6 shows a relative cell toxicity (%) of a normal conjunctival fibroblast obtained using Gd-DOTA and 2b-2. A standard deviation (SD) is obtained using a triple analysis (n=3).

(63) The cell viability for the 2b to 2e is comparable to that of Dotarem (FIG. 6). That is, when incubated for 24 h, there is no effect on cell proliferation and cell survival. There is not a substantial difference between the present series 2a to 2e in a cell viability due to the fact that any cytotoxicity does not appear for the present series 2a to 2e over a concentration range needed to acquire the signal intensity enhancement on the MR images.

(64) Embodiment 8: Kinetic Stability in Various pH Ranges

(65) The kinetic stability is expressed as evolution of a water proton relaxation rate (R.sub.1) for 2 ([Gd]=1.0 mM) in PBS under various pH conditions (pH: 1, 3, 5, 7, 9 and 11) at a room temperature, as a function of the incubation time. The measurement is executed on a 1.5 T whole body system (GE Healthcare, Milwaukee, Wis., USA).

(66) FIG. 9 shows an evolution of R.sub.1 for 2 ([Gd]=1.0 mM) in PBS as a function of an incubation time under different pH conditions.

(67) The present series exhibit high kinetic stability over a wide range of pH 3 to 11 (FIG. 9). Especially, the complex 2a exhibits sufficient kinetic stability even at pH 1.

(68) Embodiment 9: Stability Against Hydrolysis in H.sub.2O

(69) The contrast agents of the present series 100 mg are dissolved in distilled water 10 mL and is maintained as they are for 72 hours at 50 C. Using HPLC, O.D. (Optical Density) values are read at 197 nm and the stability of the agents is determined. The substances at each retention time are examined using LC Mass Spectroscopy for accurate identification of decomposed products.

(70) A following table 3 is a table showing the hydrolytic stability after 72 hours for the 2a compound. For the complex 2a, the hydrolytic stability falls because hydrolysis into the 2b (Gd-acid) occurs in 72 hours after the complex 2a is kept in water H.sub.2O. For the compound 2a, it is confirmed that cis and trans isomers thereof exist.

(71) TABLE-US-00003 TABLE 3 2a (initial) 2a (50 C./3 day) 27.11 min 97.73% 97.43% 2a (trans) 30.48 min 1.89% 1.25% 2a (cis) 2.31 min 0.01% 0.72% 2b 52.24 min 0.04% 0.19%

(72) A following table 4 is a table showing the hydrolytic stability after 72 hours for the 2b compound. For the complex 2b, increase in amount of unknown impurity occurs in 72 hours after the complex 2b is kept in water H.sub.2O. For the compound 2b, it is confirmed that cis and trans isomers thereof exist.

(73) TABLE-US-00004 TABLE 4 2b (initial) 2b (50 C./3 day) 2.38 min 92.80% 93.4% 2b (trans) 2.88 min 3.35% 2.87% 2b (cis) 3.16 min 0.30% 0.33% 3.84 min 0.35% 0.38% 7.66 min 0.12% 0.06% 24.42 min 0.06% 0.19% 28.12 min 0.56% 0.64% 29.92 min 0.09% 0.10% 34.46 min 0.34% 0.03% Acid 49.58 min 0.19% 0.21%

(74) A following table 5 is a table showing the hydrolytic stability after 72 hours for the 2c compound. For the complex 2c, very small increase in amount of unknown impurity occurs in 72 hours after the complex 2c is kept in water H.sub.2O. Thus, the 2c compound has a relatively good hydrolytic stability. For the compound 2c, it is confirmed that cis and trans isomers thereof exist.

(75) TABLE-US-00005 TABLE 5 2c (initial) 2c (50 C./3 day) 9.10 min 91.05% 93.11% 2c (trans) 10.60 min 1.35% 1.19% 2c (cis) 2.31 min 0.06% 0.11% 2b 2.58% 0.27% 0.27% 7.61% 0.14% 0.14% 19.86% 2.22% 2.30% 40.68 min 1.65% 0.002% 1c 45.53 min 0.16% 0.18% 49.81 min 0.09% 0.11%

(76) A following table 6 is a table showing the hydrolytic stability after 72 hours for the 2d compound. For the complex 2d, very small increase in amount of unknown impurity occurs in 72 hours after the complex 2d is kept in water H.sub.2O. Thus, the 2d compound has a relatively good hydrolytic stability.

(77) TABLE-US-00006 TABLE 6 2d (initial) 2d (50 C./3 day) 6.54 min 89.67% 90.10% 2d 8.86 min 0.30% 0.30% 7.79 min 0.30% 0.36% 33.15 min 1.20% 1.12% 40.33 min 0.16% 0.007% 40.81 min 0.14% 0.14% 43.77 min 0.27% 0.25% 44.51 min 0.23% 0.22% 46.56 min 0.19% 0.18%

(78) A following table 7 is a table showing the hydrolytic stability after 72 hours for the 2e compound. For the complex 2e, very small increase in amount of unknown impurity occurs in 72 hours after the complex 2e is kept in water H.sub.2O. Thus, the 2e compound has a relatively good hydrolytic stability.

(79) TABLE-US-00007 TABLE 7 2e (initial) 2e (50 C./3 day) 17.21 min 94.88% 94.66% 2e 2.32 min 0.04% 0.04% 4.98 min 0.09% 0.07% 23.70 min 1.36% 1.57% 2e isomer 40.63 min 0.03% 0.05% 42.14 min 0.01% 0.05% 43.00 min 0.05% 0.05% 44.95 min 0.05% 0.05% 45.93 min 0.04% 0.03% 46.64 min 0.02% 0.01% 48.46 min 0.03% 0.03% 49.36 min 0.18% 0.18%