Fast and easy access to efficient bifunctional chelators for MRI applications

Article abstract of DOI:10.1016/j.bmcl.2009.05.024

Novel bifunctional agents based on the AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) structure with hydroxyl, carboxyl and chloro functional groups were prepared. The Gd^III complexes show optimal magnetic properties making them

Full text of DOI:10.1016/j.bmcl.2009.05.024

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3442–3444
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fast and easy access to efficient bifunctional chelators for MRI applications
Giuseppe Gugliotta a, Mauro Botta a, Giovanni Battista Giovenzana b, Lorenzo Tei a,
*
a Dipartimento di Scienze dell’Ambiente e della Vita, Università degli Studi del Piemonte Orientale ‘A. Avogadro’, Viale T. Michel 11, 15121 Alessandria, Italy
b DiSCAFF & DFB Center, Università degli Studi del Piemonte Orientale ‘A. Avogadro’, Via Bovio 6, 28100 Novara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel bifunctional agents based on the AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic
acid) structure with hydroxyl, carboxyl and chloro functional groups were prepared. The GdIII complexes
show optimal magnetic properties making them very good candidates for conjugation to biomolecules for
molecular imaging applications. An example of conjugation to a bile acid derivative is also reported.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 9 April 2009
Revised 6 May 2009
Accepted 6 May 2009
Available online 10 May 2009
Keywords:
Molecular imaging
Magnetic resonance imaging
Gd(III) contrast agents
Bifunctional chelates
The superb anatomical resolution of Magnetic Resonance Imag-
ing (MRI) and its outstanding capacity of differentiating soft tissues
made this procedure the technique of choice in modern diagnostic
investigations. Currently, about one-third of MRI clinical scans are
carried out in the presence of Gd-contrast agents (CA) that cause
dramatic acceleration of the water proton relaxation rates, thus
providing physiological information beyond the impressive ana-
tomical resolution commonly obtained in the uncontrasted images.
The efficacy of a CA is measured by its relaxivity (r1), a parameter
defined as the increase in the 1H relaxation rate of water protons
in the presence of a 1 mM concentration of the paramagnetic com-
plex. Among the main factors influencing the relaxivity are the
number, q, of the coordinated water molecule(s) and their mean
crucial that such derivative maintain the favourable properties
of the parent complex in terms of hydration number and rate of
water exchange. Since many of the bifunctional chelating agents
used for MRI applications are based on q = 1 Gd-complexes4 and
only a few examples of q = 2 systems conjugated to biological car-
riers have been reported,5 the synthesis of AAZTA derivatives con-
taining various functional groups useful for bio-conjugation is
highly relevant for the future development of efficient molecular
imaging probes.
Two derivatives containing a differently spaced hydroxyl group
for further functionalization have been recently published: one of
them is conjugated to a lipophilic organic chain,6 while the other
is characterized as crystal structure of the corresponding GdIII com-
plex.7 The preparation of the former derivative requires seven
steps, while no synthetic details were reported for the latter. AAZ-
TA derivatives with one or two glutarate moieties replacing the
cyclic acetic pendant arms have very recently been published.8
Unfortunately, in this latter case the derivatization resulted in a
significant decrease of the residence lifetime of the coordinated
water molecules for the corresponding Gd-complexes.
We describe herein a general synthetic procedure towards a
series of AAZTA bifunctional prochelators (Scheme 1) for coupling
both to NH2-containing biomolecules and to other reactive func-
tional groups relevant for biomedical applications. The AAZTA-de-
rived chelator should provide four intact carboxylic acid functions
in addition to the cyclic diazepane ring for a stable and efficient
binding of metal ions and a function for coupling to biomolecules.
A preliminary 1H and 17O NMR relaxometric study of the GdIII com-
plex with L1 and L2 was carried out in order to compare their mag-
netic properties with those of the parent [GdAAZTA]À complex.
residence lifetime (s
M).1
The recently reported Gd-complex based on the polyamino-
carboxylate ligand AAZTA is endowed with optimal physico-chem-
ical properties for its use as MRI CA. It is characterized by high
thermodynamic stability and kinetic inertness towards transmet-
allation reactions with endogenous cations2 and shows favourable
magnetic properties associated with the presence of two inner-
sphere water molecules (q = 2) characterized by a relatively fast
water exchange rate (sM=90 ns) and a remarkable relaxivity value
of 7.1 mMÀ1 sÀ1 at 20 MHz and 298 K.3
However, in order to be of practical use in biomedical applica-
tions, a GdIII chelate characterized by an optimized relaxation effi-
ciency needs also to contain an appropriate functional group for
conjugation to specific biological vectors. At the same time, it is
* Corresponding author. Tel.: +39 0131 360208; fax: +39 0131 360250.
E-mail address: lorenzo.tei@mfn.unipmn.it (L. Tei).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.024

G. Gugliotta et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3442–3444
3443
COOH
COOH
COOH
ization is favoured by the basic environment and by the correct
orientation of the hydroxyl and carboxy groups. The protected che-
late 3 has to be stored refrigerated as it is unstable towards lacton-
ization at room temperature. The hydrolysis of the t-butyl esters by
reaction with a 50% solution of trifluoroacetic acid in CH2Cl2 re-
sulted in the formation of a mixture of L1 and of an 2-oxomorphol-
inic product by acid catalysed intramolecular Fisher
transesterification. Using less harsh conditions, for example phos-
phoric acid10 or sulphuric acid,11 the same mixture of products was
obtained although with different percentages as revealed after
HPLC-MS analysis. After treatment of the mixture with a 1 M solu-
tion of NaOH the lactone was converted quantitatively to L1 and
stored at basic pH and at T 6 5 °C. Remarkably, there are only a
few reports on intramolecular lactonization with t-butyl esters
and to the best of our knowledge only in our case the cyclization
occurs spontaneously.12
In order to form a derivative that could be easily conjugated to
biomolecules the Williamson etherification reaction was tested on
the protected ligand 3 following various reaction conditions re-
ported in the literature.13 However, the basic conditions that had
to be used resulted in the complete conversion of 3 into the 2-oxo-
morpholinic derivative 4. Hence we decided to exploit the forma-
tion of an ester bond with the hydroxyl group of 3 synthesising
the derivative (5) obtained by the opening of succinic anhydride
(Scheme 4). 5 has the great advantage that, having one free car-
boxyl group and the other protected as t-butyl esters can be used
for conjugation in organic solvents and in the classical solid phase
peptide synthesis approach, too. To validate this potential applica-
tion, the stability of the ester bond of 5 was tested in the acidic
conditions used for deprotection of the t-butyl esters (i.e., TFA/
CH2Cl2 1:1) thus obtaining the ligand L2. In addition, the protected
ligand 3 was reacted with thionyl chloride to convert the hydroxy-
methyl group into a chloromethyl derivative (6). In order to
demonstrate its versatility, we reacted 6 with 3-amino-3,7-dide-
oxycholic methyl ester14 (Scheme 4). Deprotection of both t-butyl
and methyl esters yielded the final ligand L3. On complexation
with GdIII ligand L3 gives rise to a complex which will be studied
as MRI contrast agent in comparison with other bile acid conju-
gates. In fact, several liver-specific Gd-based CAs in which a bile
acid conjugated to a Gd-DOTA or DTPA derivative have been re-
ported demonstrating their ability to target the hepatocytes and
to bind Human Serum Albumin or lipoproteins.15
COOH
COOH
COOH
N
N
N
COOH
COOH
COOH HOOC
O
HO
N
N
N
N
O
N
N
COOH
COOH
COOH
L2
COOH
AAZTA
L1
COOH
N
H
H
COOH
N
N
N
COOH
OH
L3
HOOC
Scheme 1. Ligands discussed in this Letter.
Ph
NH
NO2
(CH2O)n
HO NO2
NH2
c
Ph
a
b
+
N
HO
NH
NH
HO
NH
Ph
N
Ph
(or CH3NO2)
1
2
COOtBu
COONa
Bu
COOtBu
COOt
N
COONa
N
COONa
d,e
HO
N
N
N
HO
N
COOtBu
COONa
3
L1
Scheme 2. Synthesis of L1. Reagents and conditions: (a) Toluene/EtOH (1/1 v/v),
reflux, 6 h, purification on silica gel (99.5% from nitroethanol, 62% from nitrometh-
ane); (b) Pd/C, HCOONH4, HCOOH, MeOH, 50 °C, 1 h; ion exchange resin (97.6%); (c)
Bromo-tert-butylacetate, K2CO3, MeCN, rt, 18 h, purification on silica gel (53%); (d)
TFA/CH2Cl2, rt, 16 h; (e) NaOH 1 M, reflux,1 h.
L1 was synthesised as shown in Scheme 2 modifying the proce-
dure reported by Aime et al.3 The nitrodiazepine 1 was obtained by
double nitro-Mannich reaction between N,N’-dibenzyl ethylenedi-
amine, paraformaldehyde and 2-nitroethanol in nearly quantita-
tive yield. Alternatively
1
may be prepared by using
nitromethane and excess paraformaldehyde to form nitroethanol
in situ through the Henry reaction; in these conditions, the yield
is reduced to about 60%.
Reduction of the nitro and the N-benzyl groups is achieved by
catalytic hydrogenation on Pd/C catalyst using ammonium formate
and formic acid as source of hydrogen at T 6 50 °C. The acidic envi-
ronment and the temperature control were necessary to prevent
the formation of the alkoxide of 1 and subsequent retro-Henry
reaction.9 After removal of the formate anions by passing the
ammonium salt 2 through an anion exchange resin column, the
strongly basic amine was reacted with t-butyl bromoacetate in
the presence of anhydrous K2CO3 to generate the protected chelate
3. This alkylation was carried out in CH3CN and at room tempera-
ture in order to avoid the formation of a variable amount of a
2-oxomorpholine by-product which was formed by intramolecular
reaction between the hydroxyl group and one of the two equiva-
lent t-butyl esters on the exo-cyclic amine (Scheme 3). This lacton-
In order to check the effect of the chemical derivatization on the
relaxometric properties, the GdIII complexes of L1 and L2 were pre-
pared by mixing stoichiometric amounts of the ligands and GdCl3
solution. Unchelated Gd3+ ions were eliminated by precipitation
of the hydroxide at basic pH by adding aliquots of a concentrated
NaOH solution. Interestingly, [GdL1]À and [GdL2]À are bifunctional
COOR
COOR
COOtBu
COOtBu
N
5 - R = tBu
L2 - R = H
N
COOR
COOtBu
HOOC
O
HO
c
N
a
N
N
O
N
COOR
COOtBu
b
COOH
3
COOtBu
O
COOH
N
N
H
N
COOtBu
COOH
H
O
COOtBu
COOtBu
N
N
COOtBu
a
b
COOtBu
N
N
N
COOtBu
N
COOtBu
COOtBu
d,c,e
COOH
Cl
N
N
HO
4
N
N
COOtBu
COOH
OH
O
COOtBu
L3
HOOC
6
N
COOH
3
O
N
Scheme 4. Synthesis of functional derivatives 5, 6 and of the deprotected ligands L2
and L3. Reagents and conditions: (a) succinic anhydride, CH2Cl2, rt, 6 h (67%); (b)
thionyl chloride, CH2Cl2, rt, 2 h; (c) TFA/CH2Cl2 1:1, rt, 16 h; (d) aminocholic methyl
ester, NEt3, CH3CN, rt, 24 h; (e) NaOH 1 M, reflux 1 h; HPLC purification (26.2%
from 3).
N
COOH
Scheme 3. Intramolecular cyclization to the 2-oxomorpholine derivative: (a)
spontaneous at rt or using a base such as K2CO3 or DIPEA; (b) TFA/CH2Cl2, rt, 16 h.

3444
G. Gugliotta et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3442–3444
Gd-complexes which could exploit the free hydroxyl or carboxyl
functions for conjugation. In fact, they can be anchored in aqueous
media onto sensitive systems such as proteins which do not toler-
ate the acidic conditions necessary for the hydrolysis of the t-butyl
esters.
Supplementary data
Supplementary data (detailed synthetic procedures and spec-
troscopic data for final products and intermediates) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.05.024.
The 1H NMR relaxometric behaviour of [Gd(L1)]À and [Gd(L2)]À,
studied at 20 MHz and 298 K, does not differ markedly from the
data obtained for the parent [Gd(AAZTA)]À complex as the relaxiv-
ities are 7.6 and 9.1 mMÀ1sÀ1, respectively, and constant over a
large pH range (2–11). Also the exchange lifetimes of the two
water molecules, determined for both complexes by Nuclear Mag-
netic Resonance Dispersion (NMRD) and 1H VT NMR profiles and
confirmed by preliminary 17O NMR data, were about 90 20 ns,
in line with the value found for [Gd(AAZTA)]À.
References and notes
1. (a) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. Rev. 1999, 99,
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3. (a) Aime, S.; Calabi, L.; Cavallotti, C.; Gianolio, E.; Giovenzana, G. B.; Losi, P.;
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Giovenzana, G. B.; Palmisano, G.; Sisti, M.; Cavallotti, C.; Aime, S.; Calabi, L.
PCT Int. Appl. 2003; WO0308390.
In conclusion, the procedure for the synthesis of functionalized
AAZTA-ligands described in this Letter represents a significant ad-
vance with regard to the following:
4. (a) Woods, M.; Sherry, A. D. Chem. Today 2005, 23, 31; (b) Barge, A.; Tei, L.;
Upadhyaya, D.; Fedeli, F.; Beltrami, L.; Stefanìa, R.; Aime, S.; Cravotto, G. Org.
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(1) The number of synthetic steps is limited and analogous to
those of the parent AAZTA ligand.
(2) A large variety of functional groups can be introduced start-
ing from the protected compound 3.
(3) The conjugation to the vector molecules can be achieved
either in organic solvent (using 3, 5 and 6) or in aqueous
media (using [Gd(L1)]À or [Gd(L2)]À).
(4) The corresponding GdIII complexes maintain the favorable
relaxation properties of [Gd(AAZTA)]À.
11. Strazzolini, P.; Misuri, N.; Polese, P. Tetrahedron Lett. 2005, 46, 2075.
12. (a) Thorimbert, S.; Taillier, C.; Bareyt, S.; Humiliere, D.; Malacria, M.
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Gupta, M. Tetrahedron Lett. 2004, 45, 8825.
Then, these new compounds may have widespread utility for
the synthesis of Magnetic Resonance-Molecular Imaging probes,
not excluding the possibility to use L1 or L2 with other diagnostic
(
111In, 67/68Ga) or therapeutic (90Y, 177Lu) radiometals for nuclear
medicine applications.
14. Anelli, P. L.; Lattuada, L.; Uggeri, F. Synth. Commun. 1998, 28, 109.
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Acknowledgments
We thank the support of the Regione Piemonte (Ricerca Sani-
taria Finalizzata 2007). CIRCMSB and EC COST Action D38 are also
acknowledged.