A Novel Series of Complexones with Bis- or Biazole Structure as Mixed Ligands of Paramagnetic Contrast Agents for MRI Elena P. Mayoral, a Marı ´ a Garcı ´ a-Amo, a PilarLo´pez, a Elena Soriano, a Sebastia´n Cerda´n b and Paloma Ballesteros a, * a Departamento Quı´mica Orga ´nica y Biologı´a, Facultad de Ciencias, UNED, Senda del Rey 9, 28040 Madrid, Spain b Instituto de Investigaciones Biome ´dicas CSIC, c/ Arturo Duperier 4, 28029 Madrid, Spain Received 22 August 2002; accepted 10 July 2003 Abstract—We describe the syntheses, physicochemical properties and biological evaluation of a novel series of complexones con- taining bis- or biazoles moieties and two iminodiacetic acid units as novel ligands for paramagnetic lanthanides. The complexones were prepared by reaction of the corresponding 1,1 0 -bishaloethylbi- or bispyrazoles with methyl iminodiacetate and subsequent NaOH hydrolysis. 1,1 0 -Bisbromoethyl precursors were obtained by direct alkylation with an excess of 1,2-dibromoethane, or by heatingthecorrespondingalcoholinHCl.SigmoidalbindingisothermsandMOcalculationssupportedasmoststablestructuresin solution, those containing two Gd(III) atoms bound per molecule of complexone with half saturation values S 0.5 (M 1 ,22  C,pH 7.2) in the range 6.5 10 6 < S 0.5 < 36.1 10 6 . Relaxivity properties [r 1 , r 2 ,s 1 mM 1 Gd(III)] determined at 1.5 Tesla gave values (12.0 < r 1 < 17.7, 12.2 < r 2 < 20), improving significantly the relaxivities of reference compounds such as Gd(III)EDTA (5.2, 5.6) or Gd(III)DTPA(4.30,4.30).Theseimprovementsinvolvemainlyincreasedhydrationandslowerrotationalmotions.Invitrotoxicity experiments are reported. # 2003 Elsevier Ltd. All rights reserved. Introduction Even though Magnetic Resonance Imaging (MRI) methods inherently provide high intrinsic tissue con- trast, the use of extrinsic contrast agents has become a routine in many diagnostic imaging procedures. 1 Very frequently, the paramagnetic lanthanide Gd(III) is used to increase locally the longitudinal relaxation rate of surrounding tissue water, highlighting the intensity of specific tissue areas in T 1 weighted images. 2 However, free Gd(III) is toxic in vivo and in vitro and Gd(III) chelates must be used in the clinic for safety reasons. 3,4 ThefirstgenerationofGd(III)ligandswasderivedfrom linear polyaminopolycarboxylates such as diethylene- triaminepentaacetic acid (DTPA) or from macrocycles such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra- acetic acid (DOTA). 5 The corresponding Gd(III) com- plexes depicted very high thermodynamic and kinetic stability. 6 However, their capacity to induce water relaxation termed relaxivity, 7 remained ca. 4–5 s 1 mM 1 , well below the optimal values of approximately 100s 1 mM 1 predicted by theory. 8 Reduced relaxivity imposed the use of large doses of these agents, limiting the possibilities to visualize successfully low concen- tration molecular targets such as cell surface antigens, receptors, enzymes or even genes. 9 To overcome these limitations it became necessary to increase the relaxivity of new generations of Gd(III) chelates, maintaining simultaneously their high thermo- dynamicstability.Oneapproachtowardsthisgoal,isto maintain the basic chemical structures of Gd(III)DTPA orGd(III)DOTAcomplexesbutincreasetheirrelaxivity by restricting their rotational dynamics through con- jugation to linear polymers, dextrans and proteins or through the production of dendrimeric derivatives. 3,10 An extensive series of macromolecular DTPA and DOTA derivatives were produced and characterized in this way, increasing relaxivities to ca. 15 s 1 mM 1 per unit of bound Gd(III) chelate in linear polymers, ca. 19 s 1 mM 1 in dendrimers and ca. 50 s 1 mM 1 in those bound to serum albumin. 11 However, even these values remained below the optimal relaxivities predicted, suggesting that further improvements would 0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2003.07.002 Bioorganic & Medicinal Chemistry 11 (2003) 5555–5567 *Corresponding author. Tel.: +34-91-398-7320; fax: +34-91-398- 6697; e-mail: pballesteros@ccia.uned.es