Synthesis and biological evaluation of a new triazole-oxotechnetium complex

Article abstract of DOI:10.1039/c2ob25774b

A new triazole oxotechnetium chelating agent was synthesized via a 'Click-to-Chelate' strategy. In vivo evaluation of the corresponding ^99mTc complex shows that the tracer exhibits very interesting properties for molecular imaging.

Full text of DOI:10.1039/c2ob25774b

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PAPER
Synthesis and biological evaluation of a new triazole–oxotechnetium
complex†
Olivier Martinage, Loïc Le Clainche, Bertrand Czarny and Christophe Dugave‡*
Received 23rd April 2012, Accepted 13th June 2012
DOI: 10.1039/c2ob25774b
A new triazole oxotechnetium chelating agent was synthesized via a ‘Click-to-Chelate’ strategy. In vivo
evaluation of the corresponding 99mTc complex shows that the tracer exhibits very interesting properties
for molecular imaging.
in the stabilization of the oxotechnetium core as well. Therefore,
Introduction
we investigated the potential of triazole to chelate the oxotechne-
tium core for the preparation of biologically stable tetradentate
99mTc complexes (Fig. 1).
Technetium (99mTc)-based tracers are employed in 85% of
routine anatomic and functional radiodiagnostic exams carried
out in hospitals. Many technetium chelates have been reported in
the literature and have permitted the emergence of a wide variety
of tracers for molecular imaging. In particular, chelates contain-
ing an oxotechnetium(V) core are the most readily accessible
complexes.1
Based on previous works,3 we chose to build candidate chelat-
ing agents around a glycine central moiety bearing both a
triazole motif and a thiol group at each terminus. In fact, the
thiol group is known to facilitate oxotechnetium coordination
and consequently is found in many 99mTc complexes that exhibit
very high in vivo stabilities such as reference compounds 3 and 4
(Fig. 1).1 On the other hand, the triazole group offers a unique
potential for the development of new tracers through combinatorial
chemistry techniques.6,7
Technetium can be coordinated by heteroatoms and electron
donating chemical groups to form stable complexes.2 On the one
hand, some tetradentate oxotechnetium(V) complexes containing
either
a pyridyl (compound 1) or an imidazoyl group
(compound 2) have shown satisfactory stabilities in vivo
In this study we investigated the potential of new oxo-
technetium tetradentate chelating agents usable as novel tracers
for in vivo molecular imaging.
(Fig. 1).3
On the other hand, the study of Tc(I) chelates containing a
heterocycle such as imidazole4 and pyrazole5 has led to the
development of Tc(I)–triazole derivatives that can be obtained
through a click-chemistry strategy called ‘Click to Chelate’.6
This latter application has opened new perspectives in terms
of combinatorial chemistry and biological applications.7 To the
best of our knowledge, no data regarding the complexation of
the oxotechnetium(V) core by triazole-containing tetradentate
chelating agents could be found in the literature. The complexa-
tion of the oxorhenium core (a chemical analog of oxotechne-
tium) by benzotriazole-containing bidentate ligands was also
reported by Machura and co-workers.8 All these results
suggested that the conjugated triazole ring is able to participate
Results and discussion
Among the wide variety of oxotechnetium complexes, the N3S
chelating motif offers a unique potential in terms of possible
arrangements and substitutions.1 We explored four possible com-
binations including either a thioalkyl or a thiophenyl moiety
together with a terminal triazole group (Scheme 1). Compounds
7 and 13 feature the most adaptable structures that might facili-
tate oxotechnetium coordination. Compound 14 was anticipated
to promote rigidity of the TcO3+ square pyramidal base and to
enhance complex stability due to the combination of the
1,2,3-triazole-4-carboxyl moiety with the 1-aminothiophenyl
group.9 Compound 8 was expected to have an intermediary
rigidity. In this study, we used a simple and relatively inert
benzyl moiety as the triazole substituent. Compounds 7, 8, 13
and 14 were synthesized from acetylenic precursors using the
Huisgen [3 + 2] cyclodipolar addition7 through in situ azide sub-
stitution10 followed by acidolysis of the S-trityl group
(Scheme 1). The final products were purified by RP-HPLC and
directly used for 99mTcO3+ chelation.
CEA, iBiTecS, Service d’Ingénierie Moléculaire des Protéines,
Laboratoire de Chimie du Vivant, bâtiment 152, 91191 Gif-sur-Yvette,
France. E-mail: christophe.dugave@cea.fr; Fax: +33 16908 7991;
Tel: +33 16908 9938
†Electronic supplementary information (ESI) available: Details of exper-
imental procedures and characterization of novel compounds (46 pages).
See DOI: 10.1039/c2ob25774b
‡Present address: CEA, iBiTecS, Service de Chimie Bioorganique et de
Marquage, Laboratoire de Marquage au Tritium, bâtiment 547, 91191
Gif-sur-Yvette, France.
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Fig. 1 Chemical structures of tetradentate oxotechnetium chelating agents 1–4.
Scheme 1 General synthetic sketch of triazole-based chelating agents and corresponding Tc complexes starting from acetylenic precursors. Com-
pounds 12, 14 and 16 contain a phenyl group, which was replaced with a more flexible ethyl chain in compounds 11, 13 and 15.
led to the tetradentate chelating agent 8.10 Precursor 11 as well
as reference compounds 3 and 4 were prepared by a classical
stepwise solid-phase synthesis using a 4-methoxytrityl-cystea-
mine resin.11
Complexation of the four Tc-chelating agents was carried out
at room temperature which enabled discrimination of compounds
that do not coordinate the TcO3+ core efficiently. In three cases,
a complex mixture of radiolabelled species was eluted by
RP-HPLC. The presence of a massive peak around 5.0 min
suggested that most of the reduced technetium remained in a
non-complexed form. Increasing temperature did not improve
the Tc coordination for these compounds. Conversely, complex
10 was repeatedly obtained in quantitative yields (radio-HPLC)
and high purity (Table 1) from chelating agent 8 (Scheme 1).
The stability of complex 10 in mice plasma was investigated.
For this purpose, the complex was incubated at 37 °C and
aliquots were analyzed by RP-HPLC after varying periods of
time. As shown in Table 1 and Fig. 2, the oxotechnetium
complex 10 exhibited a stability in fresh plasma that is
Scheme 2 Example of synthesis of acetylenic precursor 6 and triazole-
containing tetradentate chelating agent 8.
equivalent to that of complex 21 with about 90% of the initial
complex remaining after one 99mTc half-life (6 h). In the same
conditions, reference complex 20 was more sensitive to plasma
constituents.
In order to evaluate the behaviour of the oxotechnetium
complex 10 in vivo, we injected a solution of this complex into
the tails of a set of four healthy female BalbC mice that were
sacrificed after 2 h. Three mice were used for tissue distribution
studies and the fourth one served for imaging. We observed a
very low accumulation of radioactivity in tissues, particularly in
liver and intestine (<5%), suggesting that, after 2 h, tracer 10 is
The acetylenic precursors 5, 6 and 12 were prepared by
classical chemical procedures using very simple reactants. As an
example, compound 6 was obtained from mercaptoacetic
acid and N-tert-butyloxycarbonylglycine as depicted in
Scheme 2 in 41% overall yield. Copper-catalyzed Huisgen
[3 + 2] cyclodipolar addition of benzyl azide, generated in situ
from benzyl bromide and sodium azide, followed by acidolysis
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Table 1 RP-HPLC retention times, ES/MS, purities and stabilities of technetium complexes 10, 20 and 21 in mice plasma at 37 °C; RP-HPLC
retention time and ES/MS of complex 22
Ligand
Complex
tR (min)a
m/zb (MALDI)
Puritya,c (%)
98
Stabilitya,d (%)
90
8
17.4
432.0b
3
11.1
14.5
8.9
—
98
99
—
60
90
—
4
—
8 + Cys
534.0e
a 99mTc complexes. b 99gTc complexes. c Radiochemical purity (RP-HPLC). d After incubation for 6 h in mice plasma at 37 °C. e MH+–H2O.
Fig. 2 Time-dependent evolution of complex 10 (%) in BalbC female
mice plasma (● complex 10, ■ complex 20, ▲ complex 21).
not metabolized through the gastro-intestinal system (Fig. 3A).
Very low radioactivity levels in blood and strong accumulation
in urine showed a massive clearance from the circulating milieu
and suggests a rapid excretion through the renal pathway. A low
but significant residual radioactivity associated with the kidneys
also supports this hypothesis. Conversely, in the same conditions,
non-complexed oxotechnetium (TcO3+) as well as the Tc complex
obtained from compound 13 mainly accumulated in liver.
Fig. 3 Tissues and fluids accumulation of complex 10 (black) and
TcO3+ (grey) injected in to female BalbC mice (22–25 g). The planar
β-imaging of complex 10 injected in to a BalbC mouse (longitudinal
section) using the minor 99mTc β-emission (E = 2 keV) showed a partial
accumulation of tracer in the kidney. A very low level of radioactivity
was detected in other tissues (inset B).
A high-resolution beta-imaging experiment using the minor 2
keV β− radiations associated with the 99mTc radioactive decay
was preferred to SPECT for these preliminary studies due to
higher resolution of the former technique. The image (Fig. 3B)
clearly shows a slight concentration of radioactivity in the cortex
of kidneys that is consistent with a predominant renal excretion
of the tracer. A very low and apparently non-specific accumu-
lation of tracer was observed throughout the whole body.
metabolization were taking place during the renal excretion. For
this purpose, we injected two supplementary mice with com-
pound 10. After 2 h, their urine was collected and analyzed by
In order to determine the behaviour of tracer 10 in vivo, we
investigated whether a tracer demetallation process12 or a partial
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transmetallation by more hydrophilic endogenous thiols such as
glutathione (GSH), cysteine or β-cysteine; a tracer metabolization
process that is frequently reported in the literature.13 In particu-
lar, GSH conjugation is known to participate in the activation
and metabolization of cis-platin derivatives.14 The RP-HPLC
elution-time of a reference obtained upon tracer/cysteine chal-
lenge (tR = 9.0 min) suggests that peak b (tR = 9.0 min) might be
the chimeric tracer–cysteine adduct 22 (Table 1). Conversely,
MALDI-MS analysis of peak c, which elutes at 11.9 min, gave
two major signals (m/z 713.3 and 743.3) that could not be ident-
ified by MS/MS fragmentation experiments. This compound was
also observed at lower levels after prolonged incubation of tracer
10 in mice plasma. This minor transformation could be related to
the binding of oxotechnetium to a plasma constituent but not to
the loading of the complex by a blood protein, in particular
serum albumin (for example through serum albumin Cys34–Tc
binding),12 since no corresponding signal could be detected. A
possible conjugation to GSH with either a partial or total dis-
sociation of complex 10 was also examined. However, isolated
compound c corresponds neither to the symmetric [bis-GS–TcO]
complex (in a native or a partially proteolyzed form) nor to the
chimeric [GS–TcO–8] complex.14
A possible metabolization of the tracer by blood erythrocytes,
as previously reported,3 was suspected. This hypothesis was
confirmed by incubation of complex 10 in fresh mice blood for
2 h at 37 °C: peak c (11.9 min) was detected as the major
product. However, this apparent sensitivity to erythrocytes was
far lower after injection in mice models. Moreover, as shown by
previous in vivo experiments, a specific tracer is usually brought
to its target in a few minutes through the vasculature, whereas
the residual radioactivity is excreted. For these reasons, the struc-
ture of the unknown 99mTc-labelled metabolite c was not further
investigated.
All these results strongly suggest that complex 10 is stable for
at least 2 h after intravenous injection in mice and exhibits an
interesting tissue distribution as a non-targeted tracer. In particu-
lar, it has a very low metabolization through the hepatic pathway.
Fig. 4 RP-HPLC radiochromatograms of pure complex 10 (A),
complex 10 incubated in mice plasma for 2 h at 37 °C (B), complex 10
incubated in mice urine for 2 h at 37 °C (C), and complex 10 recovered
from the urine 2 h after i.v. injection into BalbC mice (D).
Conclusions
radio-RP-HPLC. The tracer was recovered as a mixture of peaks
d (60%) at 17.4 min (see Fig. 4D, reference tracer 17.4 min in
Fig. 4A–4C) and peaks b and c at 9.0 (7%) and 11.9 min (33%)
respectively. In these experiments, peak a was identified as non-
complexed technetium.
In order to identify the side products b and c, the tracer was
incubated for 30 min in fresh mice urine and the incubate was
analyzed by RP-HPLC. In this experiment, the tracer was pre-
pared from a mixture of 99mTc–99gTc to enable the identification
of the products by mass spectrometry. Peaks b, c and d were iso-
lated and analyzed by MALDI/TOF mass spectrometry. The
tracer (peak d) was unambiguously identified (m/z 432.0).
Despite a rise in tracer concentration from the nanomolar to the
micromolar range, the same compounds were detected, though
compound b was obtained in a lower amount.
We identified compound 8 as a new chelating motif that com-
plexes the oxotechnetium core quantitatively at room temperature
to give the corresponding complex 10. The high stability of this
technetium complex in mice plasma for 6 h prompted us to
inject it intravenously in to a set of healthy BalbC mice in order
to investigate its behaviour in vivo. As shown from tissue distri-
bution and beta-imaging experiments, the tracer led to a remark-
ably low non-specific residual labelling of tissues. Although it is
difficult to draw conclusions regarding the general stability of
triazole-containing SN3 TcO3+ complexes, the predominant
urinary excretion pathway, confirmed by the slight labelling of
kidneys displayed by imaging slides, encourages us to investi-
gate further the potential of such imaging agents. In particular,
the triazole moiety, readily accessible from the versatile click-
chemistry strategy, offers many developments for the optimiz-
ation of new tracers by the ‘Click-to-Chelate-oxotechnetium’
combinatorial chemistry approach.
Determination of the structure of the 2 side-products was
more difficult. The elution time of peak b at 9.0 min suggested a
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77.36 (C, alkyne), 71.89 (CH, alkyne), 29.23 (N–CH2–CuCH),
28.43 (CH3, Boc); IR (KBr): 2140, 1728, 1641; ES/MS (positive
mode): 212.2 (MH+, 100%).
Experimental
General
Chemical reagents and solvents were purchased from Sigma-
Aldrich, VWR, Fluka or SDS and were of the highest purity
available. Amino acids and resins were from Novabiochem.
N-(2-Oxo-2-(prop-2-yn-1-ylamino)ethyl)-2-(tritylthio)
acet-
amide 6. Compound 18 (950 mg, 4.5 mmol) was treated with
DCM–TFA 2 : 1 (30 mL) for 1.5 h at 0 °C. After evaporation of
the solvent, toluene was chased on the product (×3). After
drying in vacuo, the residue was added to a mixture of com-
pound 17 (1.503 g, 4.5 mmol, 1.0 equiv.), DIPEA (4.7 mL,
27.0 mmol, 6.0 equiv.) and DCC (1.114 g, 5.4 mmol, 1.2 equiv.)
in DCM (20 mL) and the mixture was stirred overnight at room
temperature. After filtration of the precipitate, the filtrate was
washed with 10% citric acid (×3) and brine (×3) and the organic
layer was dried over sodium sulfate. After removal of the solvent
under reduced pressure, the resulting oil was purified by silica
gel flash chromatography (eluent: hexane–AcOEt 4 : 6) to give
compound 6 as a brown solid (0.963 g, 2.25 mmol, 50%);
1H NMR (CDCl3, 250 MHz): δ 7.27 (m, 15H, Trt), 3.99 (s, 2H,
CH2–CuC), 3.59 (s, 2H, N–CH2–CO), 3.17 (s, 2H, S–CH2–
CO), 2.15 (s, 1H, CuCH); 13C NMR (CDCl3, 62.5 MHz): δ
169.1 + 168.0 (2 CO), 143.8 (C, Trt), 129.4 + 128.2 + 128.0
(3 CH, Trt), 77.2 (CuCH), 71.8 (CuCH), 43.5 (CH2–N), 35.5
(CH2–S), 29.1 (CH2–CuC); IR (KBr): 2140, 1687, 1654;
ES/MS (positive mode) 429.1 (M + H+); Elem. anal. calcd for
C26H24N2O2S C, 72.87; H, 5.64; N, 6.54; S, 7.48; found C,
72.83; H, 5.67; N, 6.53; S, 7.45%.
[
99mTcO4]− was eluted as a physiological saline solution from
commercially available 99Mo/99mTc generator system
(ELUMATIC III, CIS bio international). Analytical RP-HPLC
purifications were achieved on a Prostar Varian chromatography
system coupled to a Varian 335 diode array detector. RP-HPLC
analyses were carried out on a Varian Pursuit C18 analytical
column (250 × 4.6 mm, 5 μm) protected by an analytical Secur-
ity Guard (Phenomenex). The chromatography system was
coupled to a gamma detector (radioflow monitor) HERM LB500
(Berthold). Preparative RP-HPLC purifications were done using
a Knauer purification system made of 2 LAPrep P310 pump
modules coupled to a LAPrep P311 UV detector and a Varian
Pursuit C18 preparative column (250 × 21.2 mm, 5 μm) or a
Varian Pursuit C18 semipreparative column (250 × 10.0 mm,
5 μm). 1H and 13C NMR spectra were recorded on Bruker
AVANCE 250 spectrometer; δ and J are reported in ppm relative
to TMS and Hz, respectively. Infrared spectra were recorded on a
JASCO FTIR-410 spectrometer. ES/MS analysis were carried
out on a Quattro Micro apparatus or on a Platform LCZ
(Micromass). MALDI TOF MS were done using an Applied
Biosystems 4800 MALDI TOF/TOF analyzer. High-Resolution
Mass Spectrometry (HRMS) was performed on a Q-tof apparatus
(Micromass). Radioactivity was counted on a Medisystem MEDI
404 gamma counter. Beta-imaging was performed on a Biospace
200 beta-imager.
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(2-(tritylthio) acet-
amido)acetamide 19. Compound 6 (214 mg, 0.5 mmol) dis-
solved in DMSO–water (9 : 1) was treated successively with
benzyl bromide (59.4 μL, 0.5 mmol, 1.0 equiv.), L-proline
(11.5 mg, 0.1 mmol, 0.2 equiv.), sodium carbonate (63.6 mg,
0.6 mmol, 1.2 equiv.), sodium ascorbate (50 μL of a 1 M
aqueous solution), CuSO4·5H2O (25 μL of a 1 M aqueous solu-
tion), and sodium azide (39.0 mg, 0.6 mmol, 1.2 equiv.). After
the solution color has turned green, the mixture was stirred over-
night at 65 °C under argon. The resulting brown mixture was
poured into iced water and stirred for 30 min. Extraction with
ethyl acetate and reduction of the volume of the organic layer
under reduced pressure gave compound 19 as a brown amor-
phous solid (107 mg, 0.19 mmol, 38%): 1H NMR (CDCl3,
250 MHz): δ 7.45 (s, 1H, CH–N–NvN), 7.39 (m, 2H, NH–
CO), 7.25 (m, 5H, CH Ar), 5.50 (s, 2H, CH2Ph), 4.51 (d, J =
5.5, 2H, CH2–CuC), 3.95 (d, J = 5.5, 2H, N–CH2–CO), 3.27
(d, J = 9.0, 2H, S–CH2–CO); 13C NMR (CDCl3, 62.5 MHz):
δ 169.01 168.0 (CO, amides), 143.7 (C, triazole), 129.4–127.8
(Ar CH, Trt + Ph), 127.0 (CH, triazole), 71.6 (C, Trt), 60.3
(CH2, Bn), 35.4 (CH2-CvC), 33.8 (N–CH2–CO), 29.0 (S–CH2–
CO); IR (KBr): 1691, 1648, 1601, 1248–1229; ES/MS (positive
mode) m/z 562.2 (M + H+, 54%), 584.2 (M + Na+, 100%),
600.1 (M + K+, 21%); Elem. anal. calcd for C33H31N5O2S C,
70.56; H, 5.56; N, 12.47; S, 5.71; found C, 70.54; H, 5.60;
N, 12.41; S, 5.69%.
2-(Tritylthio)acetic acid 17. Triphenylmethanol (5.2 g,
20.0 mmol) dissolved in TFA (60 mL) was treated with mercap-
toacetic acid (1.39 mL, 20.0 mmol, 1.0 equiv.) for 3 h at room
temperature under an atmosphere of argon. After removal of
TFA in vacuo, toluene was chased (×3) over the orange solid to
give compound 17 as a white amorphous solid (6.12 g,
18.3 mmol, 92%) which was identical to the literature data:15
1H NMR (CDCl3, 250 MHz): δ 7.31 (m, 9H, Trt), 3.05 (s, 2H,
CH2); 13C NMR (CDCl3, 62.5 MHz): δ 176.0 (CO), 143.9
(C, Ph), 129.7 (C, meta Trt), 128.2 (C, ortho Trt), 127.1
(C, para, Trt), 67.3 (C, Trt), 34.6 (CH2); ES/MS (negative
mode) 333.3 (M − H+).
tert-Butyl
(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)carbamate
18. Boc-glycine (875 mg, 5.0 mmol) was treated successively
with DIPEA (2.61 mL, 15.0 mmol, 3.0 equiv.), propargylamine
(0.320 mL, 5.0 mmol, 1.0 equiv.) and DCC (1.238 g, 6.0 mmol,
1.2 eq.) in DCM (25 mL) overnight at room temperature. After
filtration of the precipitate, the filtrate was washed with 10%
citric acid (×2) and brine (×2). The organic layer was dried over
sodium sulfate and, after filtration, the resulting oil was purified
by silica gel flash chromatography (eluent hexane–AcOEt 1 : 1)
to give compound 18 (950 mg, 4.48 mmol, 90%): 1H NMR
(CDCl3, 250 MHz): δ 6.41 (s, 1H, N–CO), 5.30 (s, 1H, N–CO),
4.08 (dd, J = 5.2, J′ = 2.5, 2H, CH2–CuC), 3.82 (d, J = 5,8,
2H, CH2–CO), 2.24 (t, J = 2.5, 1H, CuCH), 1.46 (s, 9H, Boc);
13C NMR (CDCl3, 62.5 MHz): δ 169.3 (CO), 79.28 (C, Boc),
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(2-mercapto acet-
amido)acetamide 8. Compound 19 was treated with TFA–TIPS–
water 95 : 2.5 : 2.5 for 2 h at room temperature. After removal of
the solvent in vacuo, the product was dried under reduced
pressure and dissolved in water–ACN 4 : 1. The resulting
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solution was purified by semi-preparative RP-HPLC on a semi-
preparative column Varian Pursuit C18, linear gradient 250 ×
10 mm, linear gradient (100% of 0.1% aqueous TFA to 100% of
French Animal Care and Use Committee (Décret 87-848, 87-10-
19). Mice were injected intravenously with a solution of
complex 16 (caudal injection of typically 100 μL, 8 MBq) pre-
viously filtrated on Sep-Pak Light QMA cartridge (Waters).
Another mouse was injected with the solution of oxotechnetium
alone (neutralized and filtrated as reported above) as a reference.
After 2 h, animals were anesthetized with 2% isoflurane.
ACN containing 0.1% TFA) in 30 min at 3.0 mL min−1 (tR
=
17.4 min) to give compound 8 (44 mg): analytical RP-HPLC tR
= 14.6 min, purity 99%; 1H NMR (CDCl3, 250 MHz): δ 7.45 (s,
1H, CH–N–NvN), 7.39 (m, 2H, NH–CO), 7.25 (m, 5H, CH
Ar), 5.50 (s, 2H, CH2Ph), 4.51 (d, J = 5.5, 2H, CH2–CuC),
3.95 (d, J = 5.5, 2H, N–CH2–CO), 3.27 (d, J = 9.0, 2H, S–CH2–
CO); 13C NMR (CDCl3, 62.5 MHz): δ 168.5 + 167.6 (CO),
145.1 (C-N3), 144.0, 129.1, 128.8, 128.1 (CH Ar),123.0 (CH–
N3), 65.9 (CH2 Bn), 40.0 (CH2, Gly); IR (KBr): 1684, 1664,
1651, 1224; ES/MS (positive mode) 320.1 (M + H+, 100%),
342.1 (M + Na+, 2.6%), 358.0 (M + K+, 3.6%); HRMS calcd for
C14H17N5O2S, 319.1103; found, 320.1114.
Beta-imaging studies. One mouse was anesthetized and then
immersed in an −80 °C mixture of dry ice and isopentane. After
blocking body in mounting medium, tissue sections (20 μm)
were made at −20 °C with a slicing microtome (LEICA Micro-
systems, France). Sections were kept at room temperature for 2 h
in the presence of silica gel to ensure complete drying. The
quantitative determination of the radioactivity (99mTc 2 keV
emission) in dried tissue sections and imaging were carried out
using a β-imager (Biospace Lab, Paris, France) on 3 different
sensitivities.
General protocol for 99mTcO3+ preparation and complexation.
An aliquot (20 μL, 62.5 nmol) of a 1 g L−1 solution of ligand
was successively treated with a 1 g L−1 solution of stannous
chloride (35 μL, 184.4 nmol, 3 equiv.), 0.1 M aqueous sodium
hydroxide (70 μL) and a freshly eluted solution of sodium tetra-
oxotechnetate (75 μL) in water (80 mL). After 5 min at room
temperature, the reaction was quenched by addition of 1 M
aqueous hydrogen chloride (7 μL). The complex was analyzed
by RP-HPLC using an analytical column Varian Pursuit C18
250 × 4.6 mm, linear gradient (100% of 0.1% aqueous TFA to
100% of ACN containing 0.1% TFA) in 30 min at 1.0 mL
Biodistribution studies. After 2 h, three mice (injected with
complex 10) and one mouse (injected with the solution of oxo-
technetium) were sacrificed by exsanguination after anesthesia.
Organs were removed then immediately weighed and counted
with a MEDI 404 gamma counter (Medisystem, France). The
blood and urine were removed and counted too.
Urine analysis. Aliquots of mice urine (200 μL) collected 2 h
after injection of complex 10 were acidified with 0.1 M HCl
and analyzed by analytical RP-HPLC as reported above. The
chromatogram was compared to references.
min−1
.
Preparative synthesis of 99gTc labelled complex 10. Com-
pound 8 (5 mg, 15.6 μmol) dissolved in degassed water (25 mL)
was successively treated with a solution (1 mg mL−1) of stan-
nous chloride (8.75 mL, 46.0 μmol, 3.0 equiv.), 0.1 M sodium
hydroxide (17.5 mL) and a 7.5 mM aqueous solution of
Na99gTcO4 (2.1 mL). The mixture was stirred for 5 min at room
temperature (light yellow color). The solution was neutralized by
addition of 1 M hydrogen chloride (1.75 mL). Purification of
complex 10 by semi-preparative RP-HPLC on a semi-preparative
column Varian Pursuit C18 250 × 10 mm, linear gradient (100%
of 0.1% aqueous TFA to 100% of ACN containing 0.1% TFA)
in 30 min at 3.0 mL min−1 (tR = 19.8 min) afforded 1.9 mg of
Incubation of complex 10 in mice urine. Aliquots of complex
10 (99mTc labelled enriched with 99gTc) were incubated at 37 °C
in fresh mice urine for 30 min. After acidification, the urine was
analyzed by analytical RP-HPLC. Peaks were isolated, solution
volumes were concentrated and analyzed by MALDI TOF/TOF
spectrometry.
Complex 10/cysteine challenge. 99gTc-labelled complex 10
was treated with 10 equivalent of cysteine in a pH 7.2 HEPES
buffer for 2 h at 37 °C. The mixture was analyzed by RP-HPLC
and MALDI TOF/TOF spectrometry.
1
pure complex (4.4 μmol, 28%): H NMR (CDCl3, 250 MHz):
Incubation of complex 10 in complete blood. A solution of
TriaS–99mTc (50 μL) was incubated for 2 h at 37 °C in fresh
complete blood (450 μL). After centrifugation for 15 min at
1600 rpm, the clear solution was treated with 10% TFA. After
centrifugation for 5 min at 1600 rpm, an aliquot of 50 μL diluted
in water (150 μL) was analyzed by analytical radio-HPLC.
δ 7.72 (s, 1H, NH triazole), 7.44–7.50 (m, 5H, Ph), 5.71–5.86
(m, 2H, CH2 Bn), 5.32–5.39 (d, J = 18 Hz, 1H), 4.65–4.86 (m,
3H), 4.10 (AB, JAB = 55.3 Hz, J = 17 Hz, 2H, CH2); IR (KBr):
1655, 1638, 978; ES/MS (positive mode): 431.9 (M + H+,
100%), 453.9 (M + Na+, 7.2%), 469.9 (M + K+, 1.8%).
Time-dependent stability studies of complex 10 in mice
plasma. All experiments were carried out in Eppendorf tubes
‘Low bind’. The solution of complex (50 μL, 4.92 MBq) was
added to fresh mice plasma (450 μL) at 37 °C (t = 0 min). Ali-
quots are treated with 10% TFA (150 μL). After centrifugation
for 5 min, the solution was isolated and radioactivity was
measured. Solution (50 μL) was diluted with water (150 μL) and
analyzed by analytical RP-HPLC.
Acknowledgements
We are grateful to Dr R. Thaï and Mr S. Dubois (CEA/iBiTecS/
SIMOPRO) for mass spectra analysis. We are indebted to Mrs
F. Berthon and L. Da Costa (CEA/iBiTecS/SIMOPRO) for the
preparation of fresh mice blood and plasma.
Notes and references
General protocol for in vivo studies. Four female BalbC mice
(22–25 g, Charles River Laboratories, Lyon, France) were
handled according to a protocol that has been approved by the
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