Synthesis and preliminary biological evaluation of the first 99mTc(I)-specific semi-rigid tridentate ligand based on a click chemistry strategy

Article abstract of DOI:10.1002/jlcr.3182

A novel bifunctional chelating agent based on a click chemistry strategy has been synthesized and characterized on the basis of spectroscopic techniques. The metal chelating part of this new class of tridentate N₂O ligand combined a triazole un

Full text of DOI:10.1002/jlcr.3182

Research Article
Received 29 July 2013,
Revised 25 November 2013,
Accepted 10 December 2013
Published online 14 January 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3182
Synthesis and preliminary biological
99m
evaluation of the first Tc(I)-specific
semi-rigid tridentate ligand based on
a click chemistry strategy
a
a
b,c
*
b,c**
Sihem Guizani, Nadia Malek Saied, Claude Picard, Eric Benoist,
and Mouldi Saidia
A novel bifunctional chelating agent based on a click chemistry strategy has been synthesized and characterized on the basis of
spectroscopic techniques. The metal chelating part of this new class of tridentate N2O ligand combined a triazole unit and an
aromatic ring. This latter semi-rigid framework induced a pre-organization of the chelating cavity, improving the stability of
the corresponding metallic complexes (M= 99mTc, Re). Thus, the 99mTc(CO)3 complex, obtained with good yield and excellent
radiochemical purity (>90%), exhibited a high in vitro serum stability. Tissue biodistribution in normal mice showed a rapid
clearance, no long-term retention in organs and no in vivo reoxidation of technetium-99m, making this compound a promising
99mTc-chelating system.
Keywords: bifunctional chelating agent; technetium-99m; tissue distribution; click chemistry
If a plethora of ligands ranging from bidentate to tridentate
Introduction
species have been investigated for the tricarbonyltechnetium
Among non-invasive imaging modalities, single photon computer
core, in our opinion, the most elegant and promising chemical
tomography and positron emission tomography nuclear imaging
strategy remains the click-to-chelate approach reported recently
by Schibli et al.6 Using the copper(I)-catalyzed alkyne-azide
techniques are characterized by a nanomolar range sensitivity,
which minimizes potential in vivo toxicity and allows whole-body
imaging. Although numerous radionuclides with suitable decay
properties for diagnostic applications have been used for both
nuclear techniques, technetium-99m still remains a candidate of
choice for nuclear imaging purposes. For example, it is one of the
cycloaddition reaction (CuAAC reaction), so-called click
chemistry,7 he showed that this strategy allowed the formation
of new technetium-specific chelating systems in a few steps
and with high yield, without the need for further purification,
and proceeding under friendly conditions, in aqueous media at
most used single photon computer tomography radioisotopes for
room temperature. More interestingly, he demonstrated that
labeling bioactive peptides.1 Additionally, more than 80% of
the 1,2,3-triazole moiety itself could be considered as a
radio-imaging probes are based on 99mTc-complexes, the extensive
use of this radiometal being mainly due to its ideal imaging features
(140 keV γ emitter, relatively short half life of 6.02 h) combined with
a convenient availability at low cost from the commercial 99Mo/
99mTc generator.2 Many different bifunctional chelating agents
(BCAs) allowing the formation of kinetically inert 99mTc-complexes
and their grafts to a targeting biovector have been developed.3
The structure of these chelators is largely influenced by the
oxidation states of the technetium-99m and also by the nature of
its metallic cores. Thus, if the chemistry of Tc(V) species, mainly
[TcO]3+ and [TcN]2+ cores, has firstly dominated the literature,4 an
original organometallic strategy, so-called ‘tricarbonyl core’
approach, pioneered by Schubiger’s team,5 has gained
considerable attention in the last decade. The high chemical
inertness and the ease of preparation of the water-soluble and
air-stable fac-[99mTc(CO)3(H2O)3]+ core combined with its great
availability in the form of a kit formulation (Isolink™ kit) brought
new hopes and perspectives concerning the research of
efficient 99mTc(I) complexes for molecular imaging applications.
promising chelating system for the fac-[99mTc(CO)3]+ core.6
Therefore, with the great versatility of this synthetic approach,
aRadiopharmaceutical Unit, Centre National des Sciences et Technologies
Nucléaires, Sidi Thabet 2020, Tunisia
bSPCMIB, UMR 5068, CNRS; Laboratoire de Synthèse et Physico-Chimie de
Molécules d’Intérêt Biologique, 118, route de Narbonne, F-31062, Toulouse, France
cUPS; Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt
Biologique, SPCMIB, UMR 5068, Université de Toulouse, 118, route de Narbonne,
F-31062, Toulouse, France
*Correspondence to: Sihem Guizani, Radiopharmaceutical Unit, Centre National
des Sciences et Technologies Nucléaires, Sidi Thabet 2020, Tunisia.
E-mail: sihem.guizani@gmail.com
**Correspondence to: Eric Benoist, UPS; Laboratoire de Synthèse et Physico-
Chimie de Molécules d’Intérêt Biologique, SPCMIB, UMR 5068, Université de
Toulouse, 118, route de Narbonne, F-31062 Toulouse, France.
E-mail: benoist@chimie.ups-tlse.fr
J. Label Compd. Radiopharm 2014, 57 158–163
Copyright © 2014 John Wiley & Sons, Ltd.

S. Guizani et al.
a wide range of 99mTc(I)-specific click ligands have been reported as sodium pertechnetate [Na99mTcO4] was obtained in physio-
by several research groups,8 including ours.9 Most of tridentate (or logical saline as commercial 99Mo/99mTc generator systems. Isolink
polydentate) click chelators have been built on the same model, kit™ for preparation of the 99mTc-tricarbonyl complex was donated
the chelating cavity being constituted by the 1,2,3-triazole ring by Mallinckrodt Medical B.V. (the Netherlands). Methyl azido-
and additional metal chelating arms connected at the N1 and acetate 1 was prepared as previously described.13
C4 positions of the triazole unit. It has been suggested that a
1H and 13C-NMR spectra were recorded at 300 (75.5) MHz with a
combination of an aromatic amine with an aliphatic amine and Bruker AC-300 (Bruker Corp., Billerica, MA, USA) spectrometer.
carboxylate group gave tricarbonyltechnetium complexes, which Chemical shifts are reported in parts per million, and coupling
exhibited high stability in aqueous solutions.10 Surprisingly, if constants (J) are given in Hertz (Hz). Infrared spectra were recorded
numerous 99mTc(I)-specific tridentate ligands based on a triazole on a Perkin Elmer FTIR 1725 spectrophotometer (Perkin-Elmer Inc.,
framework have been developed, to the best of our knowledge, Waltham, MA, USA) in the range 4000–400 cmÀ1. Mass spectra
none of them possess a chelating cavity including a pre-organized (desorption chemical ionization (DCI), positive mode) were
moiety. It is well-known that the rigidity of the molecular skeleton obtained on an LCT Premier Waters spectrometer (Waters Corp.,
reduces the freedom of donor atoms, and this spatial pre- MA, USA). Microanalysis was performed by the microanalytical
organization favors and stabilizes the chelate ring by an entropic department of the Laboratory of Coordination Chemistry
effect and improves the stability of the corresponding metallic (Toulouse, France). HPLC analysis was performed on an SCL-
complexes.11 Bearing that in mind, we anticipated that the 10Avp SHIMADZU HPLC system coupled to a UV-Absorbance
formation, using a two-steps click-to-chelate strategy, of a semi- detector from ICS and a Gabi gamma detector from Raytest.
rigid chelating system containing an aromatic backbone and the Separations were achieved on a reverse phase C-18 column
triazole unit, should lead in a few steps and with high yield to 250 × 4.6-mm (Shim-pack VP-ODS, SHIMADZU) eluted with a
the corresponding very stable Tc(CO)3-complex. Even though binary gradient system at a flow rate of 1 mL/min. Mobile phase
any semi-rigid ligand has yet been synthesized, the conception A was methanol containing 0.1% trifluoroacetic acid, whereas
of pre-organized chelating systems using a click-to-chelate mobile phase B was water containing 0.1% trifluoroacetic acid.
approach have been recently highlighted for the preparation of The elution profile was 0–1 min 100% B, followed by a linear
new triazole-oxotechnetium complexes.12 So in this paper, we gradient to 30% B in 10 min; this composition was held for another
described a simple, rapid, and convenient synthetic route for the 10min. After a column wash with 95% A for 5 min, the column was
first 99mTc(I)-specific semi-rigid tridentate click ligand, which re-equilibrated by applying the initial conditions (100% B) for
incorporates the design features illustrated in Scheme 1; these 15min prior to the next injection.
include the following: (i) a methyl carboxylate arm directly
connected at the N1 position of the five-membered triazole ring
as potential bioconjugation site and (ii) a N2O tridentate chelating
system combining the 1,2,3-triazole moiety with 2-aminophenol
derivative. 99mTc-radiolabelling of the ligand as well as in vitro
and in vivo behaviors in normal mice of the corresponding
99mTc-complex will be discussed.
Chemical synthesis
CAUTION : Sodium azide, when inhaled, is highly toxic.
Precautions must be taken when weighing the material such as
using a powder mask and a Teflon spatula. The azidation reactions
should be performed behind a plastic shield because of the
potential explosion.
Synthesis of 2-(prop-2-yn-1-ylamino)phenol (2)
Materials and methods
The reagent was prepared following an improved published
procedure.14
Chemicals and equipment
All purchased chemicals were of the highest purity commercially
To an ethanolic solution (70 mL) of 2-aminophenol (2.5 g,
available and used without further purification. Reactions were 23 mmol), propargyl bromide (0.5 mL, 4.5 mmol) was added over a
monitored by analytical thin layer chromatography on Merck D.C.- 40-min period. After 4 days at ambient temperature, the solution
Alufolien Kieselgel 60 F254. Chromatographic purification was was concentrated and diethylether (50 mL) was added to the
conducted using silica gel obtained from Merck. Technetium-99m residue. The precipitate was filtered off and the filtrate concentrated
Scheme 1. Synthetic routes of tridentate N2O ligand and its Re- and 99mTc-complexes. Reaction conditions: i: propargylbromide, EtOH, r.t., 4 days; ii: 1, Cu(OAc)2.H2O,
NaAsc, tBuOH/H2O, r.t., one night; iii: [Re(CO)5Cl], MeOH, 65°, one night; iv: Isolink kit™, 70°, 15 min.
J. Label Compd. Radiopharm 2014, 57 158–163
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

S. Guizani et al.
to dryness. Purification by column chromatography on silica gel estimated by HPLC. Then, the resulting Tc-complex was obtained
using pentane/AcOEt (75/25) as eluent gave the title compound in 90% yield. After RP-HPLC purification (see previous text for
as an orange powder (531 mg).
elution conditions), complex 99mTc-3 was obtained with high
Yield: 82%. 1H NMR (CDCl3): δ (ppm)= 2.25 (t, J = 2.1Hz, 1H, CH), radiochemical purity (>98%). The retention time of the Tc-complex
3.97 (d, J = 2.1Hz, 2H, CH2), 4.60 (br s, 2H, OH+ NH), 6.74–6.93 (14.9 min) is similar to that of the cold rhenium (14.2 min),
(m, 4H, HAr); MS (DCI) = 148.1 [M+ H]+, 165.1 [M+ NH4]+.
confirming the chemical identity of 99mTc-3.
Synthesis of methyl 2-[4-(2-Hydroxyphenylamino)methyl)-1H-1,2,3-
triazol-1-yl]acetate, (3)
Partition coefficient determination
Lipophilicity was studied through the partition coefficient
between n-octanol and phosphate buffer (0.125 M, pH 7.4). In a
centrifuge tube containing 500 μL of each phase, 100 μL of the
purified 99mTc complex solution was added, and the mixture
was vortexed 1 min at ambient temperature, followed by
centrifugation at 5000 rpm for 5 min. A total of 100-μL aliquots of
both buffer and organic layers were counted using a NaI well-type
gamma counter (OKIDATA, MICROLINE 320). The partition
coefficient was calculated using the formula: log P = counts in
n-octanol/counts in buffer. The reported value represents the
average of triplicate measurements.
Methyl azidoacetate 1 (437mg, 3.8mmol), alkyne derivative 2
(527 mg, 3.6mmol), copper(II) acetate monohydrate (139 mg,
0.7 mmol), and sodium ascorbate (277 mg, 1.4mmol) were mixed
in an equivolumic mixture of water/tert-butanol (50 mL) and stirred
overnight at room temperature. The resulting green solution was
diluted with ethyl acetate (40 mL) and washed twice with saturated
Na2EDTA solution (2× 40 mL). The aqueous solutions were
extracted with ethyl acetate (3× 20 mL). The organic extracts were
combined, dried over Na2SO4, and the solvent was taken off under
reduce pressure. The crude product was purified by column
chromatography on silica gel using AcOEt/pentane (60/40) as
eluent. A total of 576 mg of 3 were obtained as a green solid.
1
In vitro stability
Yield: 61%. H NMR (CD3OD): δ (ppm) = 3.77 (s, 3H, CH3), 4.46
(s, 2H, CH2), 5.22 (s, 2H, CH2), 6.53-6.71 (m, 4H, HAr), 7.85
(s, 1H, Hta); 13C-NMR (CD3OD): δ (ppm) = 40.7 (CH3), 52.9 (CH2),
54.5 (CH2), 114.0, 116.0, 120.1, 122.4 (CHAr), 126.7 (CHta), 139.2,
147.5, 149.5 (Cq), 170.1 (CO); MS (DCI) =263.2 [M+ H]+, 280.2
[M+ NH4]+.
The in vitro stability of the purified complex 99mTc-3 was
evaluated at different time points using the following procedure:
in a borosilicated vial, 99mTc-3 (100 μL) was added to 1 mL of
fresh human serum at 37 °C. Aliquots were withdrawn in duplicate
during the incubation at different time intervals till 24 h and
treated with 1 mL of acetonitrile to precipitate proteins. After
centrifugation (2000 rpm for 5 min), the supernatant was subjected
to HPLC using the same system used for quality control. Any
increase in the free pertechnetate was considered as the degree
of degradation. The complex was found to be stable.
Synthesis of the tricarbonylrhenium complex, (Re-3)
It was prepared by a substitution route from commercial [Re(CO)
5Cl]. A solution of 3 (25 mg, 95 μmol), Et3N (13.3 μL, 95 μmol), and
[Re(CO)5Cl] (38 mg, 10.5 μmol) in MeOH (3 mL) was stirred
overnight at 65 °C. After cooling to room temperature, the
solution was concentrated to dryness, and the resulting residue
purified by column chromatography on silica gel using CH2Cl2/
MeOH (95/5) as eluent. A total of 26.5 mg of Re(CO)3 complex
were obtained as a green solid.
Protein binding assay
The protein binding assay of the purified complex 99mTc-3 was
evaluated at different time points using the following procedure:
in a borosilicated vial, 99mTc-3 (100 μL) was added to 5 mL of
fresh human plasma at 37 °C. Aliquots were withdrawn during
the incubation at different time intervals till 24 h, and plasma
was separated from blood samples by centrifugation (3000 rpm
for 4 min); individual fractions were counted in a well counter.
The plasma aliquots were treated with 1 mL of acetonitrile to
precipitate proteins. After centrifugation (2000 rpm for 5 min),
the activities of both phases (supernatant and precipitate) were
measured separately. The reported value represents the average
of triplicate measurements.15
Yield: 52%. MS (DCI) = 533 [M + H]+, 561 [M + C2H5]+; IR (KBr):
ν
(NH) = 3343,
ν
(C≡O) = 2018, 1915, 1884;
ν ,
(C=O) = 1750 cmÀ1
C15H13N4O6Re: calcd (found) C, 33.90 (33.95); H, 2.47 (2.25); N,
10.54 (10.30).
Radiochemical synthesis
Preparation of fac-[99mTc (OH2)3(CO)3]+ precursor
A total of 1000 μL (about 37 MBq) of freshly eluted 99mTcO4À from
a commercial generator were added to the Isolink kit containing
sodium tetraborate (2.85 mg), sodium carbonate (7.15 mg), sodium
boranocarbonate decahydrate (2.85 mg), and sodium tartrate
dihydrate (8.5 mg). The mixture was incubated in boiling water
for 30 min. After cooling, the pH was adjusted to 2 with 1 N HCl.
Quality control of [99mTc(H2O)3(CO)3]+ precursor was performed
by reverse phase HPLC (C-18 column).
Biodistribution studies in normal mice
All experiments were carried out following the Tunisian
principles of the guide to the care and use of experimental
animals. Normal Swiss mice (males, 18–20 g) were obtained from
the Pasteur institute of Tunis. Animals were housed for 1 day
before the onset of the experiments in our laboratory housing
Synthesis of the technetium-99m complex, (99mTc-3)
A solution of ligand 3 in methanolic solution (concentration: facilities. The radiotracer 99mTc-3 (100 μL diluted in saline-EtOH
1 mg/mL) was added to an aqueous solution of the fac-[99mTc (80/20), 3.7 MBq) was injected via a lateral tail vein. At different
(H2O)3(CO)3]+ precursor. After incubation at 70 °C for 15 min, an intervals after injection, the animals (n = 3) were sacrificed by
aliquot of the reaction mixture was analyzed by HPLC to check cervical dislocation. Organs of interest, samples of blood, and
the complex formation. The labeling efficiency of the mixture muscles were collected, weighed, and counted. Bladder and
was determined by thin layer chromatography on silica gel in excreted urine were not weighed. The calculation for blood
ethyl acetate. The 99mTc-ligand purity and label stability were and muscle were based upon measured activity, sample weight,
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 158–163

S. Guizani et al.
and body composition data (considering that blood comprises the fac-[99mTc(CO)3 (H2O)3]+ with 100 μL of a solution of 3 at
7% and muscle 43% of body weight). Corrections by different 1 mg/mL concentration),
a very slight decrease of the
sample geometry were applied when necessary. Results were radiochemical yield was observed (ca. 85%). The chemical
expressed as percent dose per gram of tissue (%ID gÀ1).
identification of the radiochelate was accomplished by HPLC-
comparison of its chromatogram with that of the rhenium
analogue, as underlined in Figure 1. Similar retention times were
observed (14.9min for 99mTc-3 vs 14.2 min for Re-3), confirming
the iso-structurality of both complexes. It is noteworthy that
incubation of compound 3 with [99m Tc(H2O)3(CO)3]+ at 37°C for
Results and discussion
Chemistry
The synthesis of the tridentate N2O ligand 3 was performed in 15 min resulted in 99mTc-3 complex in 91% radiochemical yield.
three steps as described in Scheme 1. Methyl azidoacetate 1
was prepared in 63% yield, according to literature protocols.13
In vitro evaluation
The key step was the introduction of the acetylenic group on
the aromatic framework. We used an elegant and rapid route
to synthesize the 2-(prop-2-yn-1-ylamino)phenol intermediate
2.14 By stirring propargyl bromide with a large excess of
2-aminophenol in absolute ethanol during 4 days, we obtained
2 in 82%. In these conditions, the presence of the hydroxyl group
on the aromatic ring did not affect the reaction outcome, and no
O-alkylation was observed using this procedure. According to
our classical copper(I)-catalyzed alkyne-azide cycloaddition
conditions,16 compound 3 was obtained in modest yield through
the action of one equivalent of 2 and a slight excess of 1, in the
presence of the Cu(II)/sodium ascorbate catalytic system in
tBuOH/water solution. The modest yield could be explained
considering 3 as a potential copper chelating ligand. The N2O
tridentate ligand is stable at room temperature and does not
require any specialized condition for long-term storage.
The lipophilicity is an important parameter to predict and
interpret the biological activity of a molecule intended to be
injected in vivo. The lipophilic character of the radiocomplex
was assessed by the determination of the partition coefficient
(P) in physiological conditions (n-octanol/0.1 M phosphate
buffer, pH 7.4) and was expressed as log P(oct/buffer). A value
of 1.3 was found, indicating that our 99mTc complex is
moderately lipophilic.18
The complex showed excellent in vitro stability under
physiological conditions in human plasma with >99% of the
99mTc-3 compound remaining intact after 24 h. Consequently,
no reoxidation to pertechnetate was observed, even after 24 h
of incubation, as indicated by HPLC controls (Figure 2). As
expected, the Tc(CO)3 core was highly stabilized by the semi-
rigid N2O tridentate scaffold. The assays with human plasma
were also performed to determine the percentage of protein
binding. After incubation of the radiotracer in blood samples,
plasma was separated from blood samples by centrifugation
and treated with acetonitrile to precipitate the proteins. Two
collected fractions (supernatant and protein) were measured by
well gamma counter. The 99mTc-complex activity has been found
to be higher in plasma fraction compared with the blood cell
one. The protein binding study depicted low binding of the
complex with blood protein. Only 12% of the complex was
bound to serum proteins after 24 h of incubation (Figure 3).
The major part of the circulating radioactivity corresponds to
A 99mTc-complex being used in vivo in very low concentration
(10À9 M), its structural characterization is generally performed by
HPLC-comparison with the corresponding analogous rhenium
complex. Therefore, the tricarbonylrhenium complex starting
from 3 was prepared in a non-optimized synthesis. By refluxing
the ligand with a slight excess of the commercial [Re(CO)5Cl]
precursor in methanol, in the presence of triethylamine as
deprotoning agent, Re-3 was obtained in 52% yield, after silica
gel chromatography purification. Spectroscopic data were
consistent with the proposed structure of the Re-complex.
Briefly, elemental analysis and positive-ion MS spectrum are
consistent with a neutral mononuclear complex with a metal-to-
ligand ratio of 1:1. Additionally, the IR spectrum revealed firstly,
the presence of N–H stretching band of secondary aromatic amine
and secondly, three bands in the carbonyl stretching region
(2018–1884 cmÀ1), which is characteristic of a fac-octahedral
tricarbonyl metal moiety. Moreover, any OH vibrator was observed
in the IR spectrum. This proved the tridentate coordination of the
metal-tricarbonyl core via the N2O scaffold, the oxygen being on
its anionic form (OÀ) whereas the amino group remaining on its
neutral NH form. The complex is soluble in all polar organic
solvents and is stable to aerial oxidation.
99m
The
Tc-radiolabelling was performed by ligand exchange
reaction using the freshly prepared fac-[99mTc(CO)3 (H2O)3]+
intermediate as radiometal precursor, using previously
a
reported procedure.17 Several parameters, such as pH, ligand
concentration, temperature, and reaction time were optimized
to obtain the best radiolabelling yield. Thus, the best labeling
protocol involved the reaction of the fac-[99mTc(CO)3(H2O)3]+
core with 1 mL of a solution of 3 at 1 mg/mL concentration,
under acidic conditions (pH = 2), at 70 °C during only 15 min.
After HPLC purification, the 99mTc(CO)3-complex was obtained
in high yield (>90%) and with high radiochemical purity
(>98%). By working at lower concentration (radiolabelling of
Figure 1. HPLC comparison of rhenium complex Re-3 (a) and 99mTc complex
99mTc-3 (b).
J. Label Compd. Radiopharm 2014, 57 158–163
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

S. Guizani et al.
Figure 2. In vitro stability study of 99mTc-3 by HPLC at 37 °C and at different times: 30 min (up left), 1 h (up right), 2 h (bottom left), and 24 h (bottom right). This figure is
available in colour online at wileyonlinelibrary.com/journal/jlc
weight < 600, neutral species, and moderate lipophilicity) are
sufficient to observe a significant brain uptake by passive
diffusion. Concerning the latter item, it is noteworthy that a
parabolic relationship between lipophilicity and passive
diffusion over the brain–blood-barrier with an optimum for
neutral molecules having a log P(oct/buffer) between 0.5 and
2.5, was reported.18
The radiolabelled technetium compound was administrated
separately to healthy male normal Swiss mice. The quantity of
radioactivity in selected organs and blood was assessed and
the percentage of injected dose per gram of tissue at 2, 5, 30,
and 60 min post-injection, was reported in Table 1. The fast
clearance of the radiotracer from the bloodstream, reflected in
the low blood activity, indicated its high stability against
exchange reactions with blood proteins, as expected
(0.39 0.04%ID gÀ1 at 60 min). Moreover, no specific uptake or
long-term retention in organs or tissues was observed. Only a
negligible fraction of the injected radioactivity was retained by
the stomach (0.30 0.02% ID gÀ1 at 60 min) and the spleen
(0.36 0.08 % ID gÀ1 at 60 min), suggesting minimal in vivo
oxidation of technetium to pertechnetate or reduction to
colloidal technetium species.19 The higher concentration of
radioactivity was measured in liver at all times studied (13.69%
ID gÀ1 at 2 min, 12.18% ID gÀ1 at 5 min, 9.89% ID gÀ1 at 30 min,
Figure 3. Plasma protein binding study of 99mTc-3.
free complex, which is a favorable feature, in terms of its
application as an in vivo radioimaging probe.
Biodistribution studies
The biodistribution of 99mTc-3 was investigated to (i) confirm
its high stability, (ii) assess its ability to clear from nontarget
organs such as liver, intestine as well as blood, and (iii) check
if its intrinsic chemical and biological properties (molecular
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 158–163

S. Guizani et al.
Table 1. Tissue distribution data of 99mTc-3 in normal Swiss mice*
Tissues
2 min
5 min
30 min
60 min
Blood
Brain
Heart
Lungs
3.52 0.62
0.14 0.02
0.77 0.06
2.45 0.81
13.69 2.15
1.64 0.52
1.18 0.19
9.25 1.83
7.25 1.45
0.42 0.07
0.45 0.03
2.21 0.23
0.07 0.01
0.58 0.05
2.19 0.75
12.18 1.91
1.36 0.42
0.44 0.21
6.69 1.25
7.65 0.89
0.34 0.03
0.42 0.04
1.11 0.07
0.04 0.01
0.23 0.04
1.84 0.09
9.89 0.83
0.53 0.19
0.15 0.05
4.86 0.87
3.68 1.26
0.25 0.02
0.38 0.03
0.39 0.04
0.02 0.01
0.14 0.01
0.93 0.03
5.24 1.22
0.36 0.08
0.03 0.01
2.32 0.87
3.23 0.75
0.11 0.01
0.35 0.02
Liver
Spleen
Pancreas
Kidneys
Intestines
Muscle
Stomach
*Values are %ID.gÀ1 standard deviation (n = 3)
and 5.24% ID gÀ1 at 60 min). Nevertheless, although there is a
significant liver uptake at earlier post-injection times, the liver
excretion rate is relatively fast. Our compound was mainly
excreted through the hepatobiliary pathway, as evidenced by
Conflict of Interest
The authors did not report any conflict of interest.
the decreasing activity in liver and intestine. In a lesser extent, References
a
partial excretion through the renal system could be
[1] J. D. G. Correia, A. Paulo, P. D. Raposinho, I. Santos, Dalton Trans.
2011, 40, 6144.
considered. moderate residual radioactivity and fast
A
clearance associated with the kidneys supports this hypothesis.
At least, a very low accumulation of radioactivity in brain was
observed at all times studied, indicating that 99mTc-3 is not
suitable as brain imaging agent despite interesting chemical/
biological properties. Conversely, because of its promising
in vivo behavior (high stability, fast blood clearance, and non-
specific binding), the new organometallic scaffold represent
an interesting base for the development of targeted diagnostic
radiopharmaceuticals.
[2] S. Liu, S. Chakraborty, Dalton Trans. 2011, 40, 6059.
[3] (a) S. Liu, D. S. Edwards, Chem. Rev. 1999, 99, 2235; (b) S. Liu, Chem.
Soc. Rev. 2004, 33, 445; (c) G. Ribeiro Morais, A. Paulo, I. Santos,
Organometallics 2012, 31, 5693.
[4] (a) J. R. Dilworth, S. J. Parrot, Chem. Soc. Rev., 1998, 27, 43; (b) S.
Jurisson, J. D. Lydon, Chem. Rev. 1999, 99, 2205.
[5] (a) R. Alberto, R. Schibli, A. Egli, P. A. Schubiger, J. Am. Chem. Soc.
1998, 120, 7987; (b) R. Alberto, K. Ortner, N. Wheatley, R. Schibli, P.
A. Schubiger, J. Am. Chem. Soc. 2001, 123, 3135.
[6] T. L. Mindt, H. Struthers, L. Brans, T. Anguelov, C. Schweinsberg, V.
Maes, D. Tourwé, R. Schibli, J. Am. Chem. Soc. 2006, 128, 15096.
[7] (a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001,
40, 2004; (b) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless,
Angew. Chem. Int. Ed. 2002, 41, 2596.
Conclusion
In summary, a 99mTc(I)-specific semi-rigid tridentate ligand based
on a click chemistry approach was described for the first time.
The semi-rigid BCA, whose the metal chelating cavity includes
the 1,2,3-triazole moiety with the 2-aminophenol backbone,
was formed in three steps with an overall yield of 32%. The
corresponding neutral 99mTc (CO)3 complex, obtained in
excellent yield, exhibits a lipophilic character and presents
interesting in vitro and in vivo behaviors. In this context, these
promising chemical and biological features—a facile access to
the click ligand, which can be grafted to biomolecules through
the carboxylate arm, the high stability of the Tc(CO)3-complex,
which exhibits rapid clearance and no long-term retention in
organs—make this N2O tridentate ligand a promising BCA for
the development of targeted radioimaging probes. Bioconju-
gation purposes with the o-methoxyphenylpiperazine pharma-
cophore for specific 5-HT1A brain receptor targeting20 are in
progress and will be reported in another paper.
[8] (a) T. L. Mindt, C. Müller, M. Melis, M. de Jong, R. Schibli, Bioconjugate
Chem. 2008, 19, 1689; (b) H. Struthers, B. Spingler, T. L. Mindt, R.
Schibli, Chem. Eur. J. 2008, 14, 6173; (c) H. Struthers, T. L. Mindt, R.
Schibli, Dalton Trans. 2010, 39, 675; (d) C. A. Kluba, T. L. Mindt,
Molecules 2013, 18, 3206.
[9] (a) C. Camp, S. Dorbes, C. Picard, E. Benoist, Tetrahedron Lett. 2008,
49, 1979; (b) A. Seridi, M. Wolff, A. Boulay, N. Saffon, Y. Coulais, C.
Picard, B. Machura, E. Benoist, Inorg. Chem. Commun. 2011, 14, 238.
[10] R. Alberto, R. Schibli, R. Waibel, U. Abram, A. P. Schubiger, Coord.
Chem. Rev. 1999, 190–192, 901.
[11] (a) J. Le Gal, E. Benoist, M. Gressier, Y. Coulais, M. Dartiguenave,
Tetrahedron Lett. 2002, 43, 9295; (b) J. Le Gal, L. Latapie, M. Gressier,
Y. Coulais, M. Dartiguenave; E. Benoist, Org. Biomol. Chem., 2004, 2,
876; (c) J. Le Gal, F. Tisato, G. Bandoli, M. Gressier, J. Jaud, S. Michaud,
M. Dartiguenave, E. Benoist, Dalton Trans. 2005, 3800.
[12] O. Martinage, L. Le Clainche, B. Czarny, C. Dugave, Org. Biomol. Chem.
2012, 10, 6484.
[13] M. Sechi, M. Derudas, R. Dallocchio, A. Dessì, A. Bacchi, L. Sannia, F.
Carta, M. Palomba, O. Ragab, C. Chan, R. Shoemaker, S. Sei, R. Dayam,
N. Neamati, J. Med. Chem. 2004, 47, 5298.
[14] A. Manzo, A. Perboni, G. Broggini, M. Rigamonti, Synthesis 2011, 1, 127.
[15] I. Essouissi, W. Ghali, S. N. Malek, M. Saidi, Nucl. Med. Biol. 2010, 37, 821.
[16] A. Boulay, A. Seridi, C. Zedde, S. Ladeira, C. Picard, L. Maron, E.
Benoist, Eur. J. Inorg. Chem. 2010, 5058.
Acknowledgements
[17] X. Zhang, P. Zhou, J. Liu, Y. Huang, Y. Lin, Y. Chen, T. Gu, W. Yang, X.
Wang, Appl. Radiat. Isot. 2007, 65, 287.
[18] C. Fernandes, J. D. G. Correia, L. Gano, I. Santos, S. Seifert, R. Syhre, R.
Bergmann, H. Spies, Bioconjugate Chem. 2005, 16, 660.
[19] R. Visentin, M. C. Giron, M. Bello, U. Mazzi, Nucl. Med. Biol. 2004, 31, 655.
[20] L. Zimmer, D. Le Bars, J. Label. Compd. Radiopharm. 2013, 56, 105.
The authors thank Mallinckrodt Medical B.V., the Netherlands, for
Isolink kit gifts. The authors are also grateful for French
Alternative Energies and Atomic Energy Commission (CEA) for
the financial support to undertaking a fellowship program with
the SPCMIB CNRS Unit (Toulouse).
J. Label Compd. Radiopharm 2014, 57 158–163
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org