Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 65 No. 5 pp. 535ñ541, 2008

ISSN 0001-6837 Polish Pharmaceutical Society

GADOLINIUM Gd(III) COMPLEXES WITH DERIVATIVES OF NITRILOACETIC ACID: SYNTHESIS AND BIOLOGICAL PROPERTIES BOLES£AW KARWOWSKIa,b*, MICHA£ WITCZAKa, ELØBIETA MIKICIUK-OLASIKa and MA£GORZATA STUDNIAREKc Department of Pharmaceutical Biochemistry and Department of Medicinal Chemistry, b) Department of Biopharmacy, Medical University, 1 MuszyÒskiego St. 90-151, £Ûdü, Poland. c) Department of Radiology, Medical University of GdaÒsk, 3 M. Sk≥odowska-Curie St. 80-210 GdaÒsk, Poland a)

Abstract: Combinations of nitrilotriacetic acid (NTA) derivatives with technetium 99mTc are known as convenient contrasting compounds used in nuclear medicine. Because of low resolution of this method, as well as necessity for using radioactive substances we have suggested new combinations of NTA aromatic derivatives with Gd(III). The aim of the undertaken study was to obtain contrasting compounds showing high affinity to liver cells enabling performance of high-resolution imaging of this organ applying the MRI. For presented complex compounds an efficient synthesis method was developed, and in vivo biodistribution tests were performed. The obtained derivatives showed strong affinity to hepatocytes, simultaneously showing differentiation depending on substituents located within the aromatic ring. Keywords: magnetic resonance imaging, Gd(III) complex compounds with aminoaromatic derivatives of nitrilotriacetic acid, in vivo biodistribution, affinity to hepatocytes

indicates three criteria that should be met by gadolinium complexes that are to be applied as MRI contrasting compounds for liver diagnostics: 1. selective set off of unchanged, properly functioning liver cells in contrast to small changes resulting from e.g. metastatic tumor; 2. possibility of liver function level determination with special respect to dispersed changes, such as cirrhosis; 3. high resolution in bile ducts imaging together with gallbladder (8).

Magnetic resonance imaging (MRI) has been introduced as medical diagnostic method in the 70ís of the 20th century by P. Lauterbur (1). The technique is based on studies by Bloch and Purcell, who were the first to note the possibility of water protons relaxation times change through administration of paramagnetic substances (2). The first paramagnetic contrasting compound applied by Yang in imaging of alimentary system was oral ferric chloride (3), and complete diagnostic potential of contrasting compounds was presented by Carr et al. during imaging of cerebral tumors in patients following intravenous administration of [Gd(DTPA)(H2O)] (2-4). In contrast to the nuclear medicine methods, in MRI it is not necessary to use any source of ionizing radiation or introduce any radioactive compounds such as 99m Tc. Brener and Elliston showed that CT test of the whole human body in case of patients aged 45-75 may lead to increased risk of neoplastic diseases by 0.08%, and for the same age group radiation increases the risk by 2% (5). Additionally, intravenous introduction of iodine ionic compounds may cause occurrence of allergic or nephrotoxic side-effects (6). Those may, however, be minimized by administration of non-ionic contrasting compounds (7). Lauffer

MATERIALS AND METHODS Aniline, 2,6-dimethylaniline, 2,4,6-trimethylaniline and GdCl3(H2O)6 (Sigma-Aldrich), NaCl, LiOH and methanol (POCH Lublin) were used without former purification. 3-Br-2,4,6-trimethylaniline was synthesized according to Nunn et al. (9). Infrared (IR) spectra were obtained for KBr pellets on a FTIRô ATI Mattson Infinity Series. Elemental analyses were obtained on Perkin Elmer Series II CHNS/O Analyzer 2400. Mass analyses, FAB MS, were obtained on Finnigan MAT 95 (Finningan Mat GmbH) apparatus, MALDI ToF spectra were obtained on TOF-MS Voyager-Elite (PerSeptive Biosystems INC.) apparatus.

* Corresponding author: Phone: +48 42 677 91 21; Fax: +48 42 677 91 20; e-mail: [email protected]

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Imaging was performed in two minute loops during 60 min after injection of 1 mg of Gd ions (815 mg of the compounds) on MRI scanner ñ Picker Int. Eclipse 1.5T. All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments. All procedures in these experiments were approved by the Ethic Committee of the Medical Academy of GdaÒsk (Poland). GENERAL PROCEDURE Dilithium salts of compounds 1, 2, 3, 4 were obtained with good yield following the method described by Keana (10) and used for further reactions without special purification procedure. Gadolinium(III) complexes (5,6,7,8) 0.035 mol of dilithium salt of 1,2,3,4 was dissolved in 15 mL of methanol and 0.0017 mol of GdCl3(H2O)6 was added. The reaction mixture was stirred for 24 h at ambient temperature. After this time 0.0017 mol of NaCl was added as 0.1 M solution in methanol. The reaction mixture was stirred for additional 60 min to give the white crystals. The residues were filtered and then washed by methanol. The required product was isolated as white crystals. Compound 5 Molecular weight: 708,71, yield: 85%, IR (cm-1): 3403.9 (H2O), 3078.7 (Ph), 1630.6 1598.5 (C=O). MS FAB (m/z) M+: (M+ + Na) 708.0; 712.2, MS FAB (m/z) M-: (M- ñ Na) 686.2, 688.1, MALDI ToF (M-): 685, 685, 687, 689. Elemental analysis: Calc. for C24H24GdN4O10Na × (H2O)6 × (CO2)6: C 33.34, H 3.36; N 5.18 %; found: C 33.0; H 3.32; N 5.43%. Compound 6 Molecular weight: 741.83, yield: 87%, IR (cm-1): 3420.6 (H2O), 3035.1 (Ph), 1665.2, 1606.9 (C=O). MS FAB (m/z) M+: (M+ + Na) 764.3; 766.2, MS FAB (m/z) M-: (M- ñ Na) 740.1; 742.1. Elemental analysis: Calc. for C28H32GdN4NaO10 ◊ (H2O)6 ◊ (CO2)6: C 35.92; H 3.90; N 4.93%; found: C 36.21; H 5.73; N 6.13%. Compound 7 Molecular weight: 770.89, yield: 90%. IR (cm-1): 3421.8 (H2O), 3038.1 (Ph), 1632.2, 1606.9, (C=O). MS FAB (m/z) M+: (M+ + Na) 794.8; MS FAB (m/z) M-: (M- ñ Na) 770.5; 772.9. Elemental analysis: Calc. for C30H36GdN4NaO10 ◊ (H2O)6 ◊ (CO2)6: C 37.11; H 4.15; N 4.81%; found: C 37.26; H 4.41; N 5.18%. Compound 8 Molecular weight: 950.65, yield: 85%. IR (cm-1): 3436.2 (H2O); 2921.1 (Ph); 16.57.1; 1596.4(C=O). MS FAB (m/z) M+: (M++Na) 951.7; 955.8; MS FAB

(m/z) M-: (M- ñ Na) 924.9; 927.8. MALDI ToF (M-): 924.4; 929.42. Elemental analysis: Calc. for C30H34Br2GdN4NaO10 ◊ (H2O)6 ◊ (CO2)6: C 32.69 H 3.51 N 4.24%; found: C 32.31; H 3.62; N 4.17%. Magnetic resonance imaging experimental parameters: MRI method: T1-weighted FSE sequence parameters: TR = 351 ms, TE = 14 ms, FOV ñ 22 cm, BW ñ 20.8, STh ñ 5 mm/0.5 mm, Res ñ 256 ◊ 192, NSA 2, acquisition time 33 s. 6 ROI (regions of interest) were choosen: heart, liver, kidney, muscle, fat tissue, brain. Time/intensity curves were withdrawn from the measurements and relative biodistribution was calculated as percentage of total enhancement in tissues compared to the total rat enhancement. MRI sequence characteristic was tested on the phantom with different Gd concentrations and compound dose was calculated to stay in the values presenting the linear correlation of Gd concentration vs. signal intensity. RESULTS and DISCUSSION From the numerous group of elements with paramagnetic character the following ions are frequently used in MRI: gadolinium Gd3+ (11), iron Fe3+ (12) and manganese Mn2+ (13). However, due to its exceptional character of influence between Gd electrons and water protons and a high stability of combinations with ligands of DTPA (Magnevist), DOTA (Dotarem) or HOPO-TAM (14) type in physiological liquids it is Gd3+ that gained the major significance in MRI (15). Until 1999 six complexes of the metal were introduced to medical practice, and three more were clinically tested (16). Excellent paramagnetic properties of the element belonging to lanthanide series, and properties of 99mTc complexes with nitrilotriacetic acid (NTA) derivatives involving aromatic ligands, which are currently broadly used in liver and bile ducts diagnostics (17), tilted us toward attempts of NTA with Gd(III) bond synthesis. Due to specific features of gadolinium complexes it is necessary to create a 9coordinative construct in which eight coordinative bonds are occupied by groups originating from the ligand ñ organic part, and the ninth place is occupied by water molecule coordinated directly with gadolinium ion. As NTA derivatives possess only three complexing groups due to their structure (Scheme 1 and 2) thus to form a stable complex two ligands are required. The remaining three free coordinative spheres should be filled with electrons originating from three water molecules.

Gadolinium Gd(III) complexes with derivatives of nitriloacetic acid...

In order to test the possibility of contrasting compounds formation based on this strategy synthesis attempts were undertaken. Ligands for complexes, derivatives of 2,2í-(2-(R-phenyl)amino)-2-oxoethyl))diacetic acid, were obtained in reaction between properly substituted derivatives of aniline and mixed anhydride of nitrilotriacetic acid and acetic acid in pyridine (Scheme 1) (9). The resulting compounds 1, 2, 3, 4 were subjected to complex reaction with Gd2O5, according to the method described by Gris (18). However, no expected products were obtained in course of the above mentioned process. Our attention was drawn by the method of binding gadolinium with EDTA developed by

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Keana (10). Complexes 5, 6, 7, 8 (Scheme 2) were obtained as a result of the reaction of lithium salt of ligands 1, 2, 3, 4 with GdCl3(H2O)6 in methanol, at room temperature. Molar ratio of reagents was 2:1. Obtained lithium salts of the complexes were taken into the derivatives being sodium salts in the reaction with NaOH in methanol at ambient temperature, preserving the ratio reagents 1:1. Obtained products were purified by crystallization in methanol/acetonitrile system. Analysis of contrasting compounds biodistribution for the obtained complexes was performed following an intravenous administration of 8-15 mg of compound 5, 6 and 8 per rat (including about 1mg

Scheme 1. Scheme of nitrilotriacetic acid derivatives synthesis.

Scheme 2. Scheme of obtaining the Gd(III) complex compounds: i) LiOH, ii) GdCl3(H2O)6, iii) NaOH

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of Gd(III)), and studying the increase of signal intensity in T1-weighted FSE and MTC (Magnetization Transfer Contrast) (19) sequences in heart, liver, brain, fat, kidneys and muscles (Graph 1). Biological examination which had been done for the synthesized bonds showed differences in tissue biodistribution. Contrasts 6, and 8 did not cause any increase of the signal intensity for brain and heart,

probably due to the specific properties of the bloodbrain barrier and fast blood flow, whereas, following the intravenous administration of the compound 5, an increase of the heart signal was observed directly after injection. However, the signal was decreased with time, achieving the initial value after 45 min (Graph 1a). Thompson et al. (20) observed similar properties of HOPO-TAM derivatives, for which

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Graph 1. Comparison of signal intensity in MAPS sequence for heart, kidneys, liver, fat, brain, muscles before and after the intravenous administration of compound: a) 5, b) 6, c) 8.

Graph 2. Comparison of signal intensities for liver and kidneys after intravenous administration of compound a) 6 and b) 8 ñ differential curves.

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Figure 1. Result of NMR imaging of rat body before intravenous administration of compounds 6 and 8 (a) and after intravenous administration of compounds 6 and 8 (b).

growth of polyethoxyethylene chain (PEO) caused inhibition of interaction with human serum proteins (HSA). During the analysis of the studied compounds no additional influence on intensity of signals coming from fat tissue and muscles was observed. For liver, the biggest increase of the signal intensity was observed for compounds 5 and 8. Following administration of the contrast, number 5 showed up its fast accumulation in hepatocytes, with simultaneously lower concentration rise in kidneys. The concentration reaches its maximal level in liver after 17 min and in the kidneys 4-6 min p.i. With time the intensity of the signals decreased after maximum in both tissues. This may be the evidence of elimination of the contrast from the organism through bile ducts and urinary tract. Renal excretion of different compounds analyzed in the study differs too. One of the reasons for those properties of the complex 5 may be a lack of its interaction with proteins, for example with albumins contained in hepatocytes (Graph 2a). Biodistribution of compound 8 showed initial dependence similar to that of compound 5, just like as above ñ an increase of signals intensity was observed in kidneys and liver. Maximal increase for kidneys was observed after 4 min, and then, after next 4 min the level decreased to the stable value comparable to the initial one. The signal rise for liver was a surprising one. It showed increasing tendencies from the moment of administration and reached its maximal level after 58 min (Graph 2b). This behavior of the contrast agent 8 could testify for its high affinity towards liver cells

and interaction with proteins contained there, causing accumulation in hepatocytes due to strong lipophilic properties and the ionic character of that compound. CONCLUSION Undertaken attempts to synthesize new bonds of complexes 1, 2, 3, 4 with gadolinium ion Gd(III) led us to development of a highly efficient and selective method for obtaining bonds of the metal. As distinct from the previously known contrasting compounds being combinations of one organic ligand with one gadolinium ion, the compounds obtained were composed of two organic molecules combined with single gadolinium ion, Gd(III). Bonds formed by compounds 5, 6, and 8 were subjected to in vivo biodistribution tests. The tests showed different results for hepatocytes depending on the ligand located in the aromatic part of the complex. As expected, basing on the results of tests on 99mTc complex compounds used in liver diagnostics, the lowest signal amplification during MR imaging was observed in the case of compound 5 that did not contain ligands in aromatic ring. The best capture properties by hepatocytes, was shown by compound 8 showing the strongest lipophilic character. This was reflected during comparison of MR imaging of rat bodies before and after administration of compounds 6 and 8 (Figure 1). Complexes 5, 6, and 8, are potentially a group of contrasting compounds for medical diagnostics

Gadolinium Gd(III) complexes with derivatives of nitriloacetic acid...

using the nuclear magnetic resonance technique. Results of analysis of their biodistribution showed different tissue affinities depending on the structure of individual compounds. Those, however, require additional studies. Acknowledgment The support from the Ministry of Education and Science (grant 3PO5F01123) is gratefully acknowledged. REFERENCES 1. Lauterbur P.C.: Nature, 242, 190 (2003). 2. Bloch F., Hansen W.W., Packard M.: Phys. Rev., 70, 474 (1946). 3. Young I.R., Clarke G.J., Bailes D.R., Pennock J.M., Doyle F.H., Bydder G.M.: J. Comput. Tomogr. 5, 534 (1981). 4. Carr D.H., Brown J., Bydder G.M., Weinmann H.J., Speck U., Thomas D.J., Young I.R.: Lancet 1 (8375), 484 (1984). 5. Brenner D.J., Elliston C.D.: Radiology 232, 735 (2004). 6. Shellock F.G., Kanal E.: J. Magn. Reson. Imaging 10, 477 (1999). 7. Gellson T.G., Bulugahapitiya S.: Am. J. Roentgenol., 183, 1673 (2004). 8. Lauffer R.B.: Chem. Rev. 87, 901 (1987).

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