Improving the relaxivity by dimerizing Gd-AAZTA: Insights for enhancing the sensitivity of MRI contrast agents

Article abstract of DOI:10.1016/j.inoche.2010.03.014

The synthesis of a dimeric derivative of Gd-AAZTA is reported. It retains the basic properties of the parent complex (q = 2, high stability) and displays a relaxivity of 14.0 mM^-1 s^-1 at 20 MHz and 298 K. The outstanding relaxivity i

Full text of DOI:10.1016/j.inoche.2010.03.014

Inorganic Chemistry Communications 13 (2010) 663–665
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Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Improving the relaxivity by dimerizing Gd-AAZTA: Insights for enhancing the
sensitivity of MRI contrast agents
Eliana Gianolio a, Kondareddiar Ramalingam b, Bo Song b, Ferenc Kalman a, Silvio Aime a, Rolf Swenson b,
⁎
a
Dipartimento di Chimica IFM and Centro di Imaging Molecolare, Università di Torino, Via Nizza 52, 10125 Torino, Italy
b
Bracco Research USA Inc, Ernst Felder Labs, 305 College Road East, Princeton, NJ 08540, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a dimeric derivative of Gd-AAZTA is reported. It retains the basic properties of the parent
complex (q=2, high stability) and displays a relaxivity of 14.0 mM−1 s−1 at 20 MHz and 298 K. The
outstanding relaxivity is the result of the increased molecular reorientational time and of the contribution
arising from 4 to 5 water molecules in the second coordination sphere of the Gd(III) ions.
© 2010 Elsevier B.V. All rights reserved.
Received 21 January 2010
Accepted 2 March 2010
Available online 15 March 2010
Keywords:
Gadolinium chelate
MRI imaging
Relaxivity
Paramagnetic Gd(III) complexes are by far the most used contrast
agents (CA) in Magnetic Resonance Imaging. All the commercially
available systems are represented by highly stable octacoordinated
complexes containing one inner-sphere water molecule whose
exchange with the bulk water causes an overall increase in relaxation
rate of the water protons in the regions where they distribute. The
ability to act as a relaxation agent is represented by the relaxivity value
(r1p) that is defined as the relaxation enhancement brought about by
the presence of the paramagnetic complex in 1 mM concentration. The
clinically used CA display r1p of 3.7–3.8 mM−1 s−1 (0.47 T and 315 K).
The search for higher relaxivities continues to be very active as the
availability of systems (with similar biodistribution/excretion char-
acteristics of the currently used agents) that yield analogous relaxation
enhancements at significantly lower doses is, of course, an interesting
possibility. Along this line, in 2007, it was reported [1] an heptacoor-
dinated Gd(III) complex, Gd-AAZTA, that displays, at 298 K and 0.47 T,
a r1p value of 7.1 mM−1 s−1. The increase of r1p has to be ascribed to the
presence of two coordinated water molecules in the inner coordina-
tion sphere of the Gd(III) ion. Furthermore, it has been shown that Gd-
AAZTA displays a very good stability (log Kf =20.2) [2] and very
limited aptitude to transmetallation in the presence of Cu2+ and Zn2+
ions. The easy preparation of AAZTA ligand is another attractive
property and several derivatives have been reported [3–6].
This new Gd(III)-based system contains all the desirable features
of the parent Gd-AAZTA complex [1], namely two inner-sphere water
molecules in fast exchange with the bulk water, high thermodynamic
stability, no tendency to form q=0 ternary adducts with endogenous
carboxyanions, and, moreover, thanks to its dimeric nature, is
characterized by an higher relaxivity as a consequence of the increase
in the molecular reorientational time.
As for the parent AAZTA ligand, the synthesis of the dimer was carried
out through the four step reaction shown in Scheme 1. The three
component Mannich addition product 1 was obtained by the reaction of
1,5-dinitropentane, N,N-dibenzylethylenediamine, and paraformalde-
hyde in ethanol using acetic acid. Simultaneous reduction of the nitro
groups and debenzylation of 1was carried out using Pd-C 10% in methanol
to afford the amine, in 97% yield. Exhaustive alkyation of the amine with
benzyl 2-bromoacetate (K2CO3, CH3CN) furnished the proligand 3.
Deprotection of the benzyl ester (Pd-C 10%) afforded the target ligand 4
in 75% yield (complete synthesis in the Appendix). Lanthanide(III)
complexes were obtained by instantaneous reaction of stoichiometric
amounts of ligand 4 and Ln(III)Cl3 (further details in the Appendix).
The relaxivity of (Gd-AAZTA)2, measured at 25 °C and 37 °C at the
proton Larmor Frequency of 20 MHz, is 14.0 and 10.8 mM−1 s−1
respectively. The relaxivity found for (Gd-AAZTA)2 is doubled with
respect to that of the parent monomeric Gd-AAZTA, sensibly higher
than that of other reported mono-aquo dimers [7–11] and almost
comparable to that of related di-acquo dimers [12]. A detailed
relaxometric characterization of the Gd-complex has been obtained
by the measurement of the 1H-relaxivity field dependence (NMRD—
Nuclear Magnetic Resonance Dispersion profiles) at two different
temperatures and from the 17O transverse relaxation rate profile as a
function of temperature (Fig. 1). The parameters reported in Fig. 1
have been obtained by the simultaneous fitting of the three sets of
In this communication we report the synthesis, stability and
relaxometric characterization of a new dimeric derivative of the Gd
(III)-chelate with the heptadentate ligand AAZTA (Scheme 1).
⁎
Corresponding author.
E-mail address: rolf.swenson@bru.bracco.com (R. Swenson).
1387-7003/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2010.03.014

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E. Gianolio et al. / Inorganic Chemistry Communications 13 (2010) 663–665
Scheme 1. Synthesis of (AAZTA)2 ligand. Reagents and conditions: (i) acetic acid, (CH2O)n, ethanol, 80 °C, 5 h; (ii) Pd-C 10%, CH3OH, 50 psi, 24 h; (iii) Benzyl-2-bromoacetate, K2CO3,
CH3CN, 80 °C, 24 h; and (iv) Pd-C 10%, CH3OH, 50 psi, 24 h.
experimental data, using the well established Solomon-Bolember-
gen–Morgan equations [13,14] and Freed's model [15] for inner and
outer sphere contributions to the 1H-relaxivity respectively and the
Swift and Connick equations for 17O-data [16].
If compared with the parent Gd-AAZTA complex, the main
differences may be found in the values of the exchange lifetime of
the coordinated water molecules and the reorientational correlation
time, both τ2M98 and τR298 values being markedly longer with respect to
the monomeric system. Whereas lengthening of τR is expected on the
basis of the increase of the MW, the slow exchange of the coordinated
water does not appear immediately understandable. It is a matter of
fact that some of the derivatives of Gd-AAZTA (whether the structural
modification involves the arm [5] or the exocyclic carbon) display
longer τM values. However, this lengthening of the water exchange
rate is not a limiting factor on the attainable relaxivity as increasing
the temperature from 25 to 37 °C results in lower relaxivity values
over the entire range of investigated frequencies.
On the other hand, the obtained τ2R98 value exactly reflects the two-
fold increase in molecular weight passing from the parent monomeric to
the dimeric species. Interestingly, the experimental NMRD profile of (Gd-
AAZTA)2 can be satisfactory fitted only by including the contribution of
4–5 second sphere water molecules. This finding suggests that water
molecules may lie between the two halves of the ligand involved in tight
H-bonding with carboxylate oxygens from the two sets of acetic arms.
The protonation constants of the ligand and the stability constants of
the (Gd-AAZTA)2 complex were determined by pH-potentiometric
titration in 0.15 M NaCl solution at 25 °C. The pH-potentiometric titration
allowed identification of 10 pKa values (Appendix). The stability
constants of the (AAZTA)2 formed with Gd(III) ion (log KGdL =21.25,
log KGd2L =18.27) were determined at 1:1 and 2:1 metal to ligand ratio in
NaCl 0.15 M. The obtained values are in agreement with the earlier
published stability constants of the Gd-AAZTA complex measured in KCl
0.15 M2. To get a better basis for the latter comparison the stability
constants of the Gd-AAZTA complex has been re-determined in this work
using NaCl 0.15 M as ionic strength (Appendix). In fact it has been found
that log Kf is higher when measured in KCl, indicating that the AAZTA
coordination cage displays a non-negligible ability to coordinate Na+ ions.
The kinetic stability determined for the parent monomeric Gd-
AAZTA complex seems to be maintained in the dimeric system as in
vitro relaxometric assays did not show any competition effects when
Fig. 1. A — 1H NMRD profiles of 1 mM Gd-AAZTA at 25 °C (filled circles) and (Gd-
(Gd-AAZTA)2 was left for several hours in the presence of up to 10-fold
AAZTA)2 at 25 (filled squares) and 37 °C (open squares); B — 17O-R2p profiles as a
excess of ZnCl2, CaCl2 or CuCl2.
function of temperature for 12.8 mM Gd-AAZTA (filled squares) and (Gd-AAZTA)2
Replacement of the fast relaxing Gd(III) ion with the shift inducing
(open squares) recorded at 2.1 T. The principal parameters obtained from the
Yb(III) allowed us to record the high resolution 1H NMR spectrum of
simultaneous fitting of the NMRD and 17O-R2p profiles are also reported.

E. Gianolio et al. / Inorganic Chemistry Communications 13 (2010) 663–665
665
Fig. 2. 1H NMR spectra of Gd-AAZTA and (Gd-AAZTA)2 registered at 400 MHz and 298 K.
the corresponding (Yb-AAZTA)2 system (Fig. 2). As expected, the 1H
Appendix A. Supplementary data
NMR spectrum of (Yb-AAZTA)2 is quite similar to that of Yb-AAZTA:
eight equally intense resonances (integration 4) corresponding to the
eight methylenic groups of the two coordination cages and two
resonances (integration 2 and 4) corresponding to the methylenic
groups of the linker. The observed pattern is fully consistent with a
highly flexible solution structure either at the level of the acetate arms
or of the macrocyclic ring. Interestingly, the resonances of H1/H3 are
less shifted than H2 to indicate a closer spatial proximity between the
latter carbon and the Lanthanide centers. Likely, as already surmised
from the occurrence of water molecules in the second coordination
sphere, the two halves of the complex are not pushed out by the
residual negative charges. Rather the bridging water molecules
appear to keep them closely associated. To minimize the space
between the two halves the propylenic chain adopts a “slice-like”
configuration thus yielding H2 at a shorter distance than H1 and H3.
The herein reported results on (Gd-AAZTA)2 suggest that further
higher relaxivities could be obtained by designing multimeric Gd-
AAZTA-based systems as the relaxation enhancement properties of each
unit are additive and likely such multimeric derivatives may benefit
from additional second sphere contributions. The straighforward
procedure applied for the synthesis of the herein reported dimeric
ligand may be easily extended to design such multimeric derivatives.
Supporting information available: full details on the synthesis of
AAZTA dimer and on the pH-potentiometric titrations on (Gd-AAZTA)2
and Gd-AAZTA and on their respective ligands. Supplementary data
associated with this article can be found, in the online version, at doi:
10.1016/j.inoche.2010.03.014.
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Acknowledgments
Economic and scientific support from MIUR (FIRB RBNE03PX83_006,
and PRIN 2005039914 projects), the regional platform for Molecular
Imaging (PIIMDMT, “Procedure innovative di imaging molecolare per la
diagnostica e il monitoraggio terapeutico”), EC-FP6-projects MEDI-
TRANS (Targeted Delivery of Nanomedicine: NMP4-CT-2006-026668),
ENCITE (European Network for “Cell Imaging and Tracking Expertise”)
and EU-COST D38 Action is gratefully acknowledged.
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