An electroneutral macrocyclic iron(II) complex that enhances MRI contrast in vivo

Article abstract of DOI:10.1021/jm2002298

The first example of a macrocyclic ferrous complex, where two tetrazolyl pendent arms compensate the charge of the metal center, is synthesized and examined for its capacity to enhance MRI contrast in vitro and in vivo in the mouse.

Full text of DOI:10.1021/jm2002298

BRIEF ARTICLE
pubs.acs.org/jmc
An Electroneutral Macrocyclic Iron(II) Complex That Enhances
MRI Contrast in Vivo
Fayc-al Touti,† Akhilesh Kumar Singh,† Philippe Maurin,† Laurence Canaple,‡ Olivier Beuf,§
Jacques Samarut,‡ and Jens Hasserodt*,†
†Laboratoire de Chimie, UMR 5182 CNRS, Ecole Normale Superieure de Lyon, Lyon, France
‡Institut de Gꢀenomique Fonctionnelle de Lyon, IGFL UMR 5242 CNRS, ENS Lyon, INRA 1288, UCBL, Lyon, France
§CREATIS, Universitꢀe de Lyon, UMR 5220 CNRS, Inserm U1044, INSA-Lyon, Universitꢀe Lyon 1, Lyon, France
S Supporting Information
b
ABSTRACT: The first example of a macrocyclic ferrous complex, where two tetrazolyl pendent arms compensate the charge of the
metal center, is synthesized and examined for its capacity to enhance MRI contrast in vitro and in vivo in the mouse.
’ INTRODUCTION
complex 1) while ensuring the presence of two imine-type
nitrogen atoms as found in the original pyridyl groups (2).
Groups displaying these imine-type nitrogens have to exhibit a
pKa compatible with physiological pH (i.e., lower than 7.4) to
prevent weakening of the complex thermodynamically and
kinetically by protonation.12 Herein, we present the synthesis
of a novel macrocyclic chelator and its corresponding electro-
neutral iron(II) complex (1). We then report on its evaluation as
a contrast agent for T1-weighted MR imaging in vitro and in the
live animal in comparison with reference complexes 2 and 3.
We recently proposed a new approach to the design of
responsive MRI contrast agents that act in the off/on mode.
Contrary to established strategies based on the permanently
paramagnetic lanthanide ion gadolinium, our tactic makes use of
macrocyclic complexes based on iron(II) as the paramagnetic
center influencing the nuclear spins that surround the probe.1,2
Iron is a homeostatically managed metal ion in all live organisms
on earth3 that can adopt various spin states and, in its oxidation
state II, can exist in a paramagnetic (high-spin) or a diamagnetic
(low-spin) state. The macrocycle ensures high complex stability
and thus minimum leaching of the central metal ion,4,5 and
evidence has also been accumulated for its favoring the attain-
ment of the low-spin state.6 By contrast, iron(III) complexes
have already been evaluated for their capacity to act as MRI
contrast agents,7 but they cannot be made diamagnetic and thus
invisible by MRI, such as is possible for iron(II)-based agents.
We had previously identified two structurally similar iron(II)
complexes (Scheme 1, complexes 28,9 and 310,11) based on the
trivalent ligand 1,4,7-triazacyclononane (TACN).1 Complex 2
has been found to be paramagnetic in aqueous solution. It
therefore increases relaxation of surrounding water protons
and generates significant contrast in T1-weighted MR images. An
aqueous sample of complex 3 was found to be diamagnetic, and its
MR image did not show any contrast modification compared to
that of pure water. Consequently, an MRI-silent (off) or -active
(on) complex can be obtained from the base structure, depending
on the presence or absence of a third pendent arm.
’ RESULTS AND DISCUSSION
We used our very recently established strategy of introducing
the tetrazole in the form of a preformed, preprotected synthon
rather than establishing the azole in situ as is widely practiced in
the literature (see refs 15 and 16, references therein, and refs 17
and 18). This gave very satisfactory results (Scheme 2), in
contrast to those reported for a comparable synthetic target.19
We apply benzylated tetrazolylmethyl chloride15,16 (7, Scheme 2)
here for the introduction of two pendent arms by double
alkylation of formylated 1,4,7-triazacyclonane (6)20 that can be
obtained reliably from the N/N/N orthoacetal 5 by acidic
hydrolysis if great care is applied. Key intermediate 8 is purified
to homogeneity by silica gel chromatography to give satisfactory
yields (62ꢀ78%). While hydrolysis of the formamide group of 8
is straightforward (95%), the removal of the two benzyl groups
by catalytic hydrogenation requires amounts of palladium on
carbon exceeding the weight of the introduced starting material
(3.5 equiv w/w) but then succeeds efficiently in the course of 48
h at 1 atm of hydrogen. The reason for the high amounts of cata-
lyst is attributed to the presence of the tertiary amine groups,21
and our own experiences appear to indicate that bidentate
chelating motifs may also reduce the catalytic power of palladium
on carbon.15,16 We eventually ran the reaction at the gram scale,
Our first-generation paramagnetic model complex 2(Scheme1),
while generating significant relaxivity, has the fundamental dis-
advantage of carrying two positive charges. Its thermodynamic
stability is thus necessarily not optimal (lack of Coulombic
charge compensation).12 It possesses an elevated Lewis acid
character and undoubtedly an unfavorable osmolalilty. In this
context it is not surprising that commercial MRI contrast agents
based on the lanthanide ion Gd(III) are all electroneutral or
negatively charged.13,14 We set out to address these limitations
by rendering our MRI-active complex electroneutral (Scheme 1,
Received: April 4, 2011
Published: May 11, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1. Macrocyclic Ferrous Complexes
Figure 1. Average T1 values (at 7 T) of several independently synthe-
sized samples (standard deviations at column tip) at four different
concentrations (mM).
applied contrast agent [Gd(DOTA)]ꢀ1 (Dotarem, Guerbet,
France) were included. These values correspond to relaxivities
of r1 of 0.57 mMꢀ1 sꢀ1 (1) and 3.75 mMꢀ1 sꢀ1 (Dotarem). The
electronic correlation relaxation time T1e of iron(II) centers is
recognized not to be optimal for lowering T1 of the surrounding
water hydrogen spins at lower clinical field strengths (e.g., 1.5 T).1
However, at higher field strengths, T1e for iron(II) progressively
loses its role as a relevant parameter for the modulation of relaxivity
as it decreases with the square of the field.24,25 However, the water
exchange rate (a relevant parameter at high fields) of the ferrous ion
is known to be high (10ꢀ10 to 10ꢀ7 s) compared to that of the ferric
ion (>10ꢀ5 s) and in the same range as that of lanthanide ions,26
thus contributing to satisfactory relaxivity. As expected, low-spin
complex 3 modifies neither T1 nor T2 beyond a negligible factor
consistent with the temperature-independent residual paramagnet-
ism for this complex in aqueous solution (0.9 μB).1
Scheme 2a
To initially avoid the intrinsic problem of molecule deli-
very through cell membranes, we have developed an in vivo
injectionꢀelectroporation procedure into the tibialis muscle to
test molecule candidates directly in mice. The protocol was
developed at the example of the commercial contrast agent
Dotarem. Mice were treated under anesthesia (intraperitoneal
injection of ketamin (100 mg/kg) and xylazin (10 mg/kg)). The
contrast agent (50 μL, 50 mM) or pure water as negative control
was injected into the tibialis muscle and electroporated (8ꢁ,
200 V/cm, 20 ms with 500 ms of intervals) to allow diffusion into
muscle cells. MRI scans were performed 24 h postinjection. We
determined the accuracy of this internalization protocol by
verifying the degree of correlation of the MRI signal intensity
with the amount of injected gadolinium agent. The mice were
imaged using a 32 mm imaging probe and a T1-weighted spin
echo sequence in coronal and transverse orientations. The
images were acquired with TR/TE 500/12 ms and 4 NEX. For
all the scans, FOV = 30 ꢁ 30 mm2, slice thickness = 0.8 mm, and
acquisition matrix size = 256 ꢁ 192. Selected slices clearly depic-
ted the presence of contrast agent entrapped in muscle cells. By
use of homemade software, the mean value of signal intensity
within the tibialis muscle was calculated, corrected to the
experimentally determined noise standard deviation, and nor-
malized to an internal reference as shown in Figure 2A. Our
results prove that signal intensity is directly proportional to the
quantity of injected gadolinium agent.
a (a) (1) HCl, (2) NaOH, pH 11.4; 0 °C, 63ꢀ75%; (b) MeCN, TEA,
62ꢀ78%; (c) (1) NaOH, EtOH, reflux, 95%, (2) H2, 0.8 equiv of Pd/C,
60ꢀ78%; (d) Fe(BF4)2 6H2O, MeOH.
3
and subsequent purification of 9 by chromatography on a
reversed-phase cartridge led to final yields of 60ꢀ78%.
Complex 1 was synthesized by reacting ligand 9 with Fe-
(BF4)2 6H2O under argon. Elaboration of a stringent isolation/
3
purification protocol allowed for the reproducible isolation of
pure samples of 1 as a pale pink solid. We have singled out the
tetrazolylmethyl group as a pendent arm with an N-acidic moiety
to ensure electroneutrality of our target complex 1 (Scheme 1).22
It exhibits a very attractive aqueous pKa of ∼5,23 comparable to
that of acetic acid, thus offering a full negative charge at pH 7.4.
Electroneutral complex 1 is highly soluble in water and HEPES
buffer and insoluble in common organic solvents (MeOH,
CHCl3, CH2Cl2, CH3CN, THF).
T1 values (7 T, 300 MHz magnetic field strength, proton
resonance frequency) of aqueous solutions of 1 and 3 were
determined at four different concentrations (Figure 1). For com-
parison, the values for a commercial sample of the clinically
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Journal of Medicinal Chemistry
BRIEF ARTICLE
this macrocyclic ferrous complex with an N3(amine)N2-
(tetrazol)O(H2O) coordination motif and demonstrate the
efficiency of 1 in raising contrast.
’ CONCLUSION
Our results support the notion of biocompatibility of our
binary and macrocyclic ferrous complexes at the chosen doses,
whether displaying an open or closed coordination shell. They
also demonstrate that the principal two objections to the use of
iron-based MRI agents in oxidation state II, that of an unfavor-
able electronic relaxation time and only four unpaired electrons,
do not rule out their consideration for niche applications in mole-
cular and functional imaging, at least at elevated field strengths.
The here-developed synthetic methodology and the in vivo
imaging results constitute an important step toward our goal
to discover an iron(II)-based contrast agent that passes from
the low-spin (off) to the high-spin (on) state upon the
encounter of, and transformation by, a specific target enzyme
in the live animal.
Figure 2. MRI signals (mean ( SEM) averaged over at least three
independent acquisitions: as a function of Dotarem concentration
(mM) injected into the mouse tibialis, average value from at least
three animals (top); as a function of a constant Dotarem concentration
(50 μL, 0.5 mM) (bottom).
’ EXPERIMENTAL SECTION
Reagents and solvents were purchased from Aldrich, Acros, and Alfa
Aesar and used without further purification. THF was degassed and
dried of solvent columns on neutral alumina (glassware system).
Column chromatography was performed using Merck silica gel Si 60
(40ꢀ63 μm). 1H and 13C NMR spectra were recorded on a Varian Unity
500 spectrometer (499.83 and 126.7 MHz, respectively) or a Bruker
DPX 200 instrument (200.13 and 50.13 MHz, respectively). Chemical
shifts (δ) are reported in ppm (s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad) and referenced to solvent. NMR coupling
constants (J) are reported in hertz. The purity of target 1 and its
preceding ligand 9 was proved to be >95% by way of HPLC analysis.
HRMS were recorded by the “Centre Commun de Spectromꢀetrie de
Masse” of the University Claude Bernard in Lyon (France) and the
Service Central d’Analyse of the CNRS in Solaize (France).
1-Formyl-4,7-bis[1-benzyltetrazol-5-yl]methyl-1,4,7-tri-
azacyclonane (8). A mixture of 700 mg of 1-benzyltetrazol-5-
ylchloromethane 7 (3.4 mmol, 2.1 equiv) dissolved in acetonitrile
(3 mL) and distilled triethylamine (1 mL) is added to a stirred solution
of 1-carboxaldehyde-1,4,7-triazacyclononane 6 (250 mg, 1.6 mmol,
1 equiv) in 12 mL of dry acetonitrile. After 3 days a white precipitate
is formed, the solvent is removed under reduced pressure, water (5 mL)
is added, and the resulting solution is extracted with chloroform (3 ꢁ
15 mL), dried over Na2SO4, filtered, and concentrated. The crude
product is purified on a silica gel column to yield 500 mg (62%) of 8 as a
white solid. 1H NMR (CDCl3, 300 K, 200 MHz) δ: 7.93 (s, 1H); 7.32
(m, 6H); 7.13 (m, 4H); 5.57 (s, 4H); 3.82 (s, 4H); 3.29 (m, 2H); 3.01
(m, 4H); 2.85 (m, 2H); 2.59 (m, 2H); 2.44 (m, 2H). 13C NMR (CDCl3,
300 K, 50 MHz) δ: 163.6 (C4, tetrazole); 152.9 (C4, Ar); 152.6 (C4, Ar);
133.4 (CH, Ar); 133.4 (CH, Ar); 129.4 (CH, Ar); 129.2 (CH, Ar); 129.1
(CH, Ar); 127.5 (CH, Ar); 127.4 (CH, Ar); 56.3 (CH2); 54,9 (CH2);
54.2 (CH2); 53.3 (CH2); 51.1 (CH2); 51.1 (CH2); 50.1 (CH2); 50.0
(CH2); 49.9 (CH2); 46.6 (CH2).
1,4-Bis(1-benzyltetrazol-5-yl)methyl-1,4,7-triazacyclonane.
Sodium hydroxide pellets (1.4 g, 29 equiv) is added to a stirred solution
of 8 (500 mg, 0.99 mol, 1 equiv) in 10 mL of ethanol. After 24 h, 10 mL
of water is added and the aqueous phase is extracted with chloroform
(3 ꢁ 15 mL), dried over Na2SO4, filtered, and concentrated. 1,4-Bis(1-
benzyltetrazol-5-yl)methyl-1,4,7-triazacyclonane is obtained as a yellow-
ish oil (440 mg, 94%) of sufficient purity to be used in the next step as is.
1H NMR (CDCl3, 300 K, 200 MHz) δ: 7.23 (m, 6H); 7.08 (m, 4H);
5.54 (s, 4H); 3.76 (s, 4H); 2.56 (m, 4); 2.50 (m, 8H). 13C NMR (CDCl3,
Figure 3. T1-weighted images 24 h postinjection of a mouse (left) that
received 1 and 3 (25 μL of 25 mM in pure water) into its right and left
legs, respectively, by an injectionꢀelectroporation procedure into the
respective tibialis muscle, and another mouse (right) that was treated
with 1 (50 μL of 50 mM) in the left leg and pure water into the right leg.
In another experiment, we determined the kinetics of signal
disappearance (and the associated residence time) of injected
Dotarem at 24, 48, and 72 h postinjection (Figure 2B). In fact,
contrast above background can be detected up to 144 h post-
injection. The kinetics of MRI signal decrease are very small
compared to those habitually observed for intravenously admin-
istered MRI contrast agents. This result gives strong support to
the notion that our in vivo injectionꢀelectroporation protocol
internalizes molecular contrast agents and that it is a valid
method for testing our molecule candidates directly in mice,
even in the absence of any chemical/molecular delivery strategy.
We then applied this administration protocol to our test
candidates 1 and 3 and the imaging of the thus treated mice
(Figure 3). Samples of 1 (varying from 25 μL of 25 mM to 50 μL
of 50 mM) raised T1 contrast to a significant degree (2.7-fold
over background), while the low-spin and diamagnetic ferrous
complex 3 did not modify contrast to any extent. Several repeti-
tions of the above experiments with independently prepared
samples of complex 1 attest to the in vivo tolerance observed for
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BRIEF ARTICLE
300 K, 50 MHz) δ: 152.9 (C4, tetrazole); 133.6 (C4, Ar); 129.2 (CH,
Ar); 128.9 (CH, Ar); 127.5 (CH, Ar); 53.2 (CH2); 53.1 (CH2); 51.0
(CH2); 49.5 (CH2); 47.3 (CH2).
1,4-(Bis(tetrazol-5-yl)methyl)-1,4,7-triazacyclonane (9). In
our hands, optimization of the hydrogenation reaction was possible only
by use of LCMS. It is particularly important to advance the reaction as
much as possible in no more than 48 h and by using a new commercial
batch of Pd/C to avoid the presence of residual, partially deprotected
polyamines that are difficult to separate.
’ ABBREVIATIONS USED
MRI, magnetic resonance imaging; TACN, 1,4,7-triazacyclono-
nane; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid); DOTA, 1,4,7,10-tetraazacyclododecane; TR/TE, repeti-
tion time and echo time; NEX, number of excitations; FOV, field
of view
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been determined to be in excess of 95%; refer to the Supporting
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’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra of synthetic in-
b
termediates and tested complex 1; HPLC results and mass
spectra of 9 and 1; MRI protocol. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
*Phone: þ33 (0) 472 72 8394. Fax: þ33 (0) 472 72 8860.
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’ ACKNOWLEDGMENT
Technical assistance in the synthesis of the TACN system by
Delphine Pitrat and financial support from the National Cancer
Institute (INCA, Projet Libres 2007, via Canceropole CLARA) is
acknowledged.
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