Preparation and preliminary bioevaluation of a 99mTc(CO) 3-glucose derivative prepared by a click chemistry route

Article abstract of DOI:10.1002/jlcr.2933

Development of a ^99mTc-labelled glucose derivative as a single-photon emission computed tomography analogue to [¹⁸F]-2- fluoro-2-deoxy-D-glucose (FDG) is considered of great interest. Herein, we present the synthesis and preliminary bioevaluation of a ^99mTc(CO) ₃-glucose derivative. Derivatization of glucose at C2 was achieved using the so-called 'click chemistry', forming a histidine-like, 1,4-disubstituted triazole adequate as donor atom system for Tc(I)-tricarbonyl complexes. Synthesis of the intermediate azide derivative was achieved by conventional organic chemistry. Because of the efficiency and selectivity of this cycloaddition, labelling was performed in 'one pot', adding the tricarbonyl precursor fac-[^99mTc(CO)₃(H₂O) ₃]⁺ to the same vial where the click reaction has taken place without any further purification. A single product with radiochemical purity higher than 90% was obtained. It was stable for at least 4 h in reaction milieu and exhibited high hidrophilicity (log P of -1.2) and low binding to plasma proteins (5 ± 1%). Biodistribution in C57BL/6 mice bearing induced Lewis murine lung carcinoma is characterized by low blood and liver uptake and rapid urinary excretion. ^99mTc complex showed moderate tumour uptake but significant retention until 2 h post-injection. Soft tissue clearance was fast leading to significantly higher uptake in tumour in comparison to muscle (p = 0.05) at all time points. Overall, biodistribution of our compound was very similar to that of [¹⁸F]-FDG. However, tumour uptake was significantly higher for [¹⁸F]-FDG, probably because of high hidrophilicity of our derivative that may hinder cell penetration. Similarity to biodistribution of FDG is a promising outcome, and modifications of the chelator and linker might improve biological results. Copyright

Full text of DOI:10.1002/jlcr.2933

Research Article
Received 20 March 2012,
Revised 18 April 2012,
Accepted 23 April 2012
Published online 24 May 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2933
Preparation and preliminary bioevaluation of
a 99mTc(CO)3-glucose derivative prepared by a
click chemistry route
Soledad Fernández,a Nancy Crócamo,a Marcelo Incerti,b Javier Giglio,a
b
a
*
Laura Scarone, and Ana Rey
Development of a 99mTc-labelled glucose derivative as a single-photon emission computed tomography analogue to [18F]-
2-fluoro-2-deoxy-D-glucose (FDG) is considered of great interest. Herein, we present the synthesis and preliminary bioevaluation
of a 99mTc(CO)3-glucose derivative. Derivatization of glucose at C2 was achieved using the so-called ‘click chemistry’, forming a
histidine-like, 1,4-disubstituted triazole adequate as donor atom system for Tc(I)-tricarbonyl complexes. Synthesis of the inter-
mediate azide derivative was achieved by conventional organic chemistry. Because of the efficiency and selectivity of this
cycloaddition, labelling was performed in ‘one pot’, adding the tricarbonyl precursor fac-[99mTc(CO)3(H2O)3]+ to the same vial
where the click reaction has taken place without any further purification. A single product with radiochemical purity higher than
90% was obtained. It was stable for at least 4 h in reaction milieu and exhibited high hidrophilicity (log P of À1.2) and low bind-
ing to plasma proteins (5Æ 1%). Biodistribution in C57BL/6 mice bearing induced Lewis murine lung carcinoma is characterized
by low blood and liver uptake and rapid urinary excretion. 99mTc complex showed moderate tumour uptake but significant
retention until 2 h post-injection. Soft tissue clearance was fast leading to significantly higher uptake in tumour in comparison
to muscle (p = 0.05) at all time points. Overall, biodistribution of our compound was very similar to that of [18F]-FDG. However,
tumour uptake was significantly higher for [18F]-FDG, probably because of high hidrophilicity of our derivative that may hinder
cell penetration. Similarity to biodistribution of FDG is a promising outcome, and modifications of the chelator and linker might
improve biological results.
Keywords: Tc-tricarbonyl complexes; glucose derivative; ‘click chemistry’; tumour imaging agent
biomolecules,8–10 we present herein the preparation and
Introduction
preliminary bioevaluation as potential radiopharmaceutical for
nuclear oncology of a 99mTc(CO)3-glucose derivative. Labelling
Glucose is considered as a key molecule in metabolic evaluation
of tumours, because a nearly universal property of primary and
was performed using the pendent approach in order to preserve
metastatic cancer is the upregulation of glycolysis, which can
the biological activity. Derivatization of glucose was achieved
result in increased glucose consumption.1 Therefore, availability
using the so-called ‘click chemistry’, a Huisgen’s reaction, which
of a 99mTc-labelled glucose derivative as a single-photon emis-
occurs through a Cu(I)-catalyzed [3 + 2] cycloaddition of alkynes
sion computed tomography analogue to the well-established
and azides forming a histidine-like, 1,4-disubstituted triazole
positron emission tomography tracer [18F]-2-fluoro-2-deoxy-D-
adequate as donor atom system for Tc(I)-tricarbonyl complexes
(Figure 1).11–15
glucose (FDG) is considered of great interest. Apart from the
low costs and wide availability of 99mTc, the analogy in chemistry
Biodistribution studies in C57 mice bearing tumours induced
between technetium and rhenium, together with the fact
by inoculation of 3LL cells of Lewis murine carcinoma are also
that radioisotopes 186 and 188 of rhenium have appropriate
presented.
nuclear properties for therapy (186Re, t1/2 = 90.6 h, Ebmáx. = 1.1MeV,
Eg = 137keV; 188Re, t1/2 = 17 h, Ebmáx. = 2.1 MeV, Eg = 155 keV), con-
tribute to this preference, because the studies of 99mTc complexes
may be further expanded to the preparation of analogous rhenium
compounds for therapy.
aCátedra de Radioquímica, Facultad de Química, Universidad de la República,
Montevideo, Uruguay
Although several groups have reported the preparation of
99mTc-labelled glucose and glucosamine analogues, to our
bCátedra de Química Farmacéutica, Facultad de Química, Universidad de la
República, Montevideo, Uruguay
knowledge, none of them has yet the expected properties, and
further research in this area is still necessary.2–7
As part of our ongoing research in the application of new
*Correspondence to: Ana Rey, Facultad de Química, Universidad de la República,
General Flores 2124, 11800 Montevideo, Uruguay.
99mTc moieties, namely Tc(V)-nitride, 4+1 mixed ligand Tc(III)
and Tc(I)-tricarbonyl complexes, to the labelling of small
E-mail: arey@fq.edu.uy
J. Label Compd. Radiopharm 2012, 55 274–280
Copyright © 2012 John Wiley & Sons, Ltd.

S. Fernández et al.
HO
The desired chelating system was introduced by means of the
Huisgen’s [3 + 2] cycloaddition, one of the most representative
examples of ‘click chemistry’. It consists of an innovative functio-
nalization strategy for biomolecules because it is efficient, selec-
tive and devoid of side reactions. The mild reaction conditions
are well suited for the modification of biomolecules, into which
the required azide or alkyne functionalities were previously
incorporated by standard synthetic transformations.11 In addi-
tion, 1,4-disubstituted 1,2,3-triazoles share structural and electro-
nic features with 1,4-disubstituted imidazoles of Ne-derivatized
histidines, which have been shown to be extraordinarily good
chelators for organometallic cores of technetium.12 Figure 3
depicts procedures used for deprotection and derivatization by
‘click chemistry’ of the glucose derivative containing an azide
group (compound 3).
O
HO
OH
CO2H
NH2
O
HO
N
N
N
Figure 1. Glucose derivative bearing a 1,4 disubstituted triazole as donor system
for 99mTc labelling.
Results and discussion
This methodology has been applied recently by E. Benoist
et al.20 to the synthesis of C1 functionalized glucose derivatives
containing various tridentate chelating systems. They have
prepared an interesting series of 99mTc and Re complexes with
promising biodistribution in normal animals. Another interesting
aspect is that they demonstrated for the first time the possibility
to graft a pre-chelated M(CO)3 core directly onto a biomolecule
by click chemistry. The reported data open new possibilities in
the development of 99mTc-labelled glucose derivatives.
Our approach, however, was to take advantage of the
high efficiency and selectivity of this cycloaddition, and conse-
quently labelling was performed in ‘one pot’, directly adding
the tricarbonyl precursor fac-[99mTc(CO)3(H2O)3]+ obtained with
radiochemical purity >90% (either by reduction of pertechnetate
in the presence of CO(g) or by use of a lyophilized kit16,21) to the
same vial where the click reaction has taken place without any
separation or purification. A single product with a retention time
of 14 min and radiochemical purity higher than 90% was
obtained, as demonstrated by HPLC analysis. The proposed
structure of the 99mTc complex is shown in Figure 4.
As a first step to achieve our goal, synthesis of a glucose deriva-
tive containing adequate donor groups for 99mTc labelling using
the Tc-tricarbonyl core was pursued (Figure 1). The synthesis
route used for the intermediate azide preparation is shown in
Figure 2.
b-D-Pentaacetyl glucose was used as starting material; the
anomeric carbon was protected using a piperidine group.
According to the extensive study performed by R. Schibli
et al.,17 we decided to introduce a bromobutyl chain linker in
C2 position by a two-step procedure. The first step involved
the anomeric C protection with piperidine and selective hydroly-
sis of C2 to afford compound 1 by precipitation.18 The following
step consisted in a nucleophilic substitution with 1,4-dibromo
butyl to obtain compound 2 in moderate yield.19 Then, substitu-
tion of the bromine atom employing trimethylsylil azide allows
us to obtain azide group necessary for the [3 + 2] cycloaddition
in good yield. All intermediate products were obtained in rea-
sonable yield and high purity as demonstrated by 1H-NMR analy-
sis. Elimination of the protective acetate groups of the hexose
should be performed in order to obtain the final product. How-
ever, purification of the deprotected glucose derivative to elimi-
nate salts that co-precipitated in the process is very difficult
because of its high polarity. Consequently, we decided to
perform deprotection in situ before each labelling without
further purification.
Stability for more than 4 h in reaction milieu was also
determined by HPLC analysis.
The partition coefficient between 1-octanol and phosphate
buffer pH 7.0 of the technetium complex was measured in order
to assess their lipophilicity. A log P of À1.2 was obtained, indicat-
ing high hydrophilicity.
Figure 2. Synthesis route for the preparation of azide derivative of glucose (compound 3).
J. Label Compd. Radiopharm 2012, 55 274–280
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

S. Fernández et al.
Figure 3. Elimination of protective groups of glucose derivative 3 and introduction of donor system by ‘click chemistry’.
HO
and biodistribution studies were performed 20 to 30 days after
inoculation when tumours have adequate size.
O
Table 1 summarizes the results expressed as % dose/organ in
the most significant organs as a function of time.
HO
OH
Results are in full agreement with physicochemical properties of
the 99mTc complex. Biodistribution is characterized by low blood
and liver uptake. Excretion occurs rapidly through the urinary tract
as expected for a compound with low lipophilicity. Thyroid and
stomach activities are very low indicating minimal in vivo reoxida-
tion. Uptake in other organs was negligible. Uptake in tumour is
moderate at all time points, probably because of high hydrophili-
city that may hinder cell penetration.
O
HO
O
H2N
99mTc
CO
N
O
N
N
CO
OC
Table 2 summarizes in vivo tumour uptake expressed as %
dose/g as well as tumour/muscle ratio.
99mTc complex demonstrated moderate tumour uptake
(0.49 Æ 0.03% dose/g at 0.5 h post-injection) and significant reten-
tion until 2 h (0.35 Æ 0.01% dose/g). Soft tissue clearance is fast,
Figure 4. Proposed structure of 99mTc-labelled glucose derivative.
Binding to plasma proteins was studied using size exclusion and this was reflected in tumour/muscle ratios, which were very
chromatography. Ideally, low protein binding is required in favourable in the studied period (1.8 Æ 0.1 at 0.5 h to 2.75 Æ 0.06 at
order to ensure adequate pharmacokinetics of the potential 2 h). Statistical analysis demonstrated that uptake in tumour was sig-
radiopharmaceuticals. Additionally, only the unbound fraction nificantly higher in comparison to muscle (p=0.05) at all time points.
of the radiotracer will penetrate cells and other biological mem-
Although the uptake of various 99mTc-labelled glucose deriva-
branes.22 A relatively low protein binding of 5 Æ 1% was tives in tumour have been reported earlier, their comparison is
obtained, correlating with the high in vitro stability and low very difficult because of the heterogeneity of the type of tumours
lipophilicity of this complex.
used. However, analysis of previously reported data5–7,23 showed
Glucose derivative bearing the azide group (3) and that tumour uptake in most of them is low, in the same order of
propargylglicine were also labelled with 99mTc and analysed by 99mTc-labelled glucose derivative 5, specially at longer retention
HPLC. Substitution was above 90% when propargylglicine was times and even those that have higher tumour uptake3,24 have
used as ligand, but HPLC revealed three major peaks with longer low tumour/blood ratio.
retention times than our labelled glucose derivative. On the
In order to compare with the gold standard, biodistribution
other hand, when compound
3
was used as ligand, of [18F]-FDG (provided by CUDIM, Uruguayan Centre for
substitution of tricarbonyl precursor was negligible. Molecular Imaging) in the same animal model and the same
Radiochromatograms of all labelled species are shown in experimental conditions was also performed. Results are shown
Figure 5.
In order to assess the potentiality of our approach for the
in Tables 3 and 4.
Although the overall biodistribution of our compound is very
design of a potential radiopharmaceutical for nuclear oncology, similar to that of [18F]-FDG and tumour/muscle ratios at longer
the biodistribution of the 99mTc complex was evaluated at biodistribution times are also comparable, the total tumour
30 min, 1 h and 2 h post-injection in C57BL/6 mice bearing uptake and the tumour dose/gramme are significantly higher
tumours induced by inoculation of 3LL Lewis murine lung carci- for [18F]-FDG, probably because of high hidrophilicity that
noma cells. Cells were inoculated subcutaneously in the right limb, may hinder cell penetration. Although 18F-FDG is even more
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 274–280

S. Fernández et al.
99m Tc-Tricarbonylic precursor
99m Tc-glucose derivative
35000
30000
25000
20000
15000
10000
5000
200000
150000
100000
50000
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
99m Tc-labelled propargyl glicine
99m Tc- labelled azide derivative of glucose
50000
40000
30000
20000
10000
0
70000
60000
50000
40000
30000
20000
10000
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
Figure 5. Radiochromatograms of various stages of 99mTc labelling of glucose derivatives.
Table 1. Biodistribution results of 99mTc(CO)3-glucose derivative in C57BL/6 bearing induced Lewis murine lung carcinoma
% Injected dose
Organ
0.5 h
1.0 h
2.0 h
Blood
Liver
2.15 Æ 0.15
8.07 Æ 0.38
2.08 Æ 0.28
0.08 Æ 0.05
2.57 Æ 0.81
0.20 Æ 0.06
1.94 Æ 0.70
10.94 Æ 2.19
67.65 Æ 0.01
2.06 Æ 0.43
5.61 Æ 0.65
0.93 Æ 0.23
0.12 Æ 0.07
2.19 Æ 0.74
0.25 Æ 0.12
1.29 Æ 0.55
16.30 Æ 5.73
73.10 Æ 5.10
0.82 Æ 0.09
3.02 Æ 0.44
0.47 Æ 0.04
0.10 Æ 0.02
0.83 Æ 0.09
0.10 Æ 0.07
0.98 Æ 0.08
16.88 Æ 4.25
70.20 Æ 3.10
Kidneys
Thyroid
Muscle
Tumour
Stomach
Intestine
Bladder + urine
Each value represents the mean percentage (n = 3) of the injected dose/organ of tissue, Æstandard deviation of the mean.
and not by passive diffusion. To the best of our knowledge, there
Table 2. Uptake in tumour of 99mTc(CO)3-glucose derivative
is no experimental evidence that any of the 99mTc-labelled
glucose derivatives developed so far can be recognized by the
in C57BL/6 bearing induced Lewis murine lung carcinoma
% Injected dose/gramme
transporter, and consequently, cell penetration might be due to
passive diffusion.
Organ
0.5 h
1.38 Æ 0.10
1.0 h
2.0 h
Blood
Muscle
Tumour
Tumour/muscle
1.45 Æ 0.35
0.70 Æ 0.20
0.13 Æ 0.05
0.35 Æ 0.01
2.75 Æ 0.06
0.27 Æ 0.08 0.26 Æ0.10
Experimental
0.49 Æ 0.03
1.80 Æ 0.11
0.55 Æ 0.05
2.13 Æ 0.15
General
All laboratory chemicals were reagent grade and were used without
further purification. Solvents for chromatographic analysis were HPLC
grade. [99mTc]NaTcO4 was obtained from a commercial generator
(Tecnonuclear S.A., Buenos Aires, Argentina). Carbonyl labelling agent
Isolink was provided by Covidien (Dublin, Ireland). NMR spectra were
obtained in the indicated deuterated solvent using a Bruker Avance
DPX 400 Spectrometer (Massachusetts, USA). Chemical shifts are
reported as d values (parts per million) relative to residual protons of
Each value represents the mean percentage (n = 3) of the
injected dose/g of wet tissue, Æstandard deviation of the
mean.
hydrophilic than 99mTc-labelled glucose derivative
P = À2.21 determined using the same experimental conditions),
5 (log
FDG is taken up by cells through the specific transporter GLUT-1 deuterated solvent. Coupling constants are reported in Hertz (Hz). The
J. Label Compd. Radiopharm 2012, 55 274–280
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

S. Fernández et al.
Table 3. Biodistribution results of [18F]-FDG in C57BL/6 bearing induced Lewis murine lung carcinoma
% Injected dose
Organ
0.5 h
1.0 h
2.0 h
Blood
Liver
5.52 Æ 0.48
5.07 Æ 0.54
1.67 Æ 0.06
0.15 Æ 0.04
18.32 Æ 0.11
17.32 Æ 0.18
1.31 Æ 0.09
7.09 Æ 0.91
22.70 Æ 3.87
2.98 Æ 0.18
3.21 Æ 0.06
0.95 Æ 0.15
0.24 Æ 0.07
27.74 Æ 1.89
19.72 Æ 3.29
0.78 Æ 0.01
8.09 Æ 0.58
6.85 Æ 0.49
2.03 Æ 0.69
3.05 Æ 0.08
0.84 Æ 0.18
0.13 Æ 0.03
24.62 Æ 4.75
18.75 Æ 4.05
1.40 Æ 0.05
6.42 Æ 0.70
5.62 Æ 0.70
Kidneys
Thyroid
Muscle
Tumour
Stomach
Intestine
Bladder + urine
Each value represents the mean percentage (n = 3) of the injected dose/organ of tissue, Æstandard deviation of the mean.
2-CH), 3.62–3.56 (m, 1H, 5-CH), 2.93–2.86 (m, 2H, CH2–N), 2.64–2.56
Table 4. Uptake in tumour of [18F]-FDG in C57BL/6 bearing
(m, 2H, CH2–N), 2.06 (s, 6H, 2x CH3), 2.01 (s, 3H, CH3), 1.62–1.44 (m, 6H,
induced Lewis murine lung carcinoma
3xCH2).
% Injected dose/gramme
N-(3,4,6-Tri-O-acetyl-2-O-(6-bromobutyl)b-D-glucopyranosyl)
piperidine (2)
Organ
0.5 h
1.0 h
2.0 h
Compound 1 (2 g, 5.35 mmol) was dissolved in benzene (50 mL) at room
temperature. Dibromobutane (1.77 g, 8.20 mmol), molecular sieves (5 g),
Ag2CO3 (3.7 g) and AgClO4 (1.66 g) were added. The reaction mixture
was heated to 50–55 ꢀC and allowed to react protected from light for
5 days. After reaching room temperature, the precipitate was filtrated
through celite and rinsed with benzene. Benzene was evaporated in
vacuo, and the residue was purified by flash column chromatography
(petroleum ether : ethyl acetate 2:1). Yield: 40% 1H-NMR (400 MHz, CDCl3):
d: 5.03 (t, 1H, 3-CH), 4.95 (t, 1H, 4-CH), 4.20 (dd, 1H, 6-CH2), 4.01 (dd, 1H,
6-CH2), 3.94–3.85 (m, 2H, 1-CH, 2-CH), 3.85 (t, 2H, CH2N3), 3.54–3.47
(m, 1H, 5-CH), 3.41 (dt, 2H, CH2O), 2.90–2.85 (m, 2H, CH2N), 2.68–2.63
(m, 2H, CH2N), 2.07 (d, 6H, 2xCH3), 2.01 (s, 3H, CH3), 1.92–1.81 (m, 2H,
CH2), 1.70–1.40 (m, 12H, 6xCH2).
Blood
Muscle
Tumour
Tumour/muscle
3.85 Æ 0.06
1.96 Æ 0.08
1.64 Æ 0.12
2.92 Æ 0.42
8.19 Æ 1.39
2.80 Æ 0.31
1.99 Æ 0.02 2.84 Æ0.30
8.04 Æ 0.06
4.04 Æ 0.02
7.95 Æ 0.87
2.80 Æ 0.21
Each value represents the mean percentage (n = 3) of the
injected dose/g of wet tissue, Æ standard deviation of the
mean.
multiplicity is defined by s (singlet), t (triplet) or m (multiplet). HPLC
analysis was developed on an LC-10 AS Shimadzu Liquid Chromatography
System (Kyoto, Japan) using a reverse phase column Phenomenex Luna
5 mm, C18 column (4.6Â 150 mm). Elution was performed with a binary
gradient system at 1.0mL/min flow rate using triethylamine-phosphate
buffer pH 2.5 as mobile phase A and methanol as mobile phase B; the
elution profile was as follows: 0 to 3 min 100% A; 3 to 6 min linear gradient
to 25% B; 6 to 9 min linear gradient to 34% B; 9 to 20 min linear gradient
to 100% B; 20 to 27 min 100% B; and 27 to 30 min linear gradient 0% B.
Detection was accomplished either with a photodiode array detector
(SPD-M10A, Shimadzu) that recorded UV–vis spectra on flux or with a
‘3 Â 3’ NaI (Tl) crystal scintillation detector. Activity measurements
were performed either in a Dose Calibrator, Capintec (Ramsey, NJ, USA)
CRC-5R or in a scintillation counter, ‘3 Â 3’ NaI (Tl) crystal detector
associated to an ORTEC monochannel analyzer.
(2R,3S,4R,5R)-2-(Acetoxymethyl)-5-(4-azidobuthyl)-6-(piperidin-1-
yl)-tetrahydro-2H-pyran-3,4-diyl diacetate (3)
To a stirred solution of 2 (2.0 g, 4.06 mmol) in dry dimethylformamide
(30 mL), trimethyl-sililazide (805 mg, 7.00 mmol) and NH4Cl (400 mg,
7.00 mmol) were added at room temperature, and the suspension was
stirred at 115 ꢀC under nitrogen for 4 h. The solution was poured onto
ice water and extracted with Et2O. The organic layer was washed with
brine, dried over MgSO4 and evaporated in vacuo. The residue was
purified with a silica column using petroleum ether : ethyl acetate (1:1)
as eluent, to yield compound 3. Yield: 65%. 1H-NMR (400 MHz, CDCl3):
d: 5.03 (t, 1H, 3-CH), 4.95(t, 1H, 4-CH), 4.20 (dd, 1H, 6-CH2), 4.01 (dd, 1H,
6-CH2), 3.94–3.85 (m, 2H, 1-CH, 2-CH), 3.85 (t, 2H, CH2N3), 3.54–3.47
(m, 1H, 5-CH), 3.41 (dt, 2H, CH2O), 2.90–2.85 (m, 2H, CH2N), 2.68–2.63
(m, 2H, CH2N), 2.07 (d, 6H, 2xCH3), 2.01 (s, 3H, CH3), 1.92–1.81 (m, 2H, CH2),
1.70–1.40 (m, 12H, 6xCH2).
Synthesis
Radiolabelling
N-(3,4,6-Tri-O-acetyl-b-D-glucopyranosyl)piperidine (1)
Deprotection
b-D-Pentaacetyl glucose (20 g, 0.05 mol) was cooled to 20 ꢀC, and
piperidine (23 mL, 0.23 mol) was added slowly. The reaction was kept at
20–25 ꢀC for 30 min until the suspended solution became clear, and after
Compound 3 (22 mg, 4.68 mmol) was dissolved in acetone–water
solution (2:1) (4.5mL) and glacial acetic acid (1.0mL, 1.00 mmol) was added.
15 min, precipitation began. The reaction was cooled to 0 ꢀC, and After the solution was stirred for 1h at 50 ꢀC, methanol was added
diethylether (80 mL) was added. After 4 h stirring, the reaction mixture (3.76 mL), and the reaction mixture was neutralized with a lithium hydro-
was allowed to reach room temperature and filtered. The solid was rinsed xide solution (0.5M, 2 mL). The mixture was stirred overnight, and the
with diethylether and cold ethanol and dried ‘in vacuo’. Yield: 39%. solvent was evaporated under reduced pressure. The residue containing
1H-NMR (400 MHz, CDCl3): d: 5.11 (t, 1H, 4-CH), 4.94 (t, 1H, 3-CH), 4.21
(dd, 1H, 6-CH2), 4.08 (dd, 1H, 6-CH2), 3.87 (d, 1H, 1-CH), 3.69 (t, 1H,
compound 4 (Figure 3) was dissolved in methanol and used for labelling
without further purification.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 274–280

S. Fernández et al.
(0.2 mL each) from each layer were counted in a gamma counter. The
partition coefficient was calculated as the mean value of each cpm/mL
of 1-octanol layer divided by that of the buffer. Lipophilicity was
expressed as log P.
Preparation of triazole derivative of glucose derivative (5)
A solution of propargylglycine in water (1.131 mg/mL, 50.0mL) was mixed
with a solution of the deprotection mixture in methanol (containing
1.286 mg/mL of compound 4, 65.0 mL). Copper acetate solution in water
(1.996mg/mL, 7.5mL) and sodium ascorbate in water (1.980 mg/mL,
15.0 mL) were added and the mixture heated for 1 h at 75 ꢀC. After cooling,
the reaction mixture was diluted with PBS buffer (300mL).
Animal studies
All animal studies were approved by the Ethics Committee of the Faculty
of Chemistry from Uruguay.
Preparation of fac-[99mTc(OH2)3(CO)3]+ complex
Using a kit formulation
Biodistribution in animals bearing induced tumours
99mTc-sodium pertechnetate (185–1850 MBq, 1 mL) was added to an
Isolink kit (Covidien, USA) and the mixture incubated at 100ꢀC for
30min. After cooling, pH was adjusted to 7.0 with 1N HCl solution. Com-
plex formation was checked by HPLC analysis as indicated in the general
experimental section. Retention time: 4.0min. Radiochemical purity> 90%.
A culture of 3LL Lewis murine lung carcinoma cells was expanded and
treated with trypsine previous to inoculation. A cell suspension in PBS
containing 3 Â 106 cells was prepared and injected subcutaneously in
the right limb of C57BL/6 mice (8–10 weeks old). The animals developed
palpable tumour nodules 20–30 days later (1.5 Â 0.5 Â 0.5 cm) and were
used for biodistribution studies.
Using CO (g)
Three animals per group were injected via a lateral tail with either
99mTc compound or [18F]-FDG (0.1 mL, 0.037–0.37 MBq). At different
intervals after injection, the animals were sacrificed by neck dislocation.
Whole tumour and samples of blood and muscle were collected,
weighed and assayed for radioactivity. Results were expressed as %
dose/organ and % dose/g tissue.
The 99mTc-precursor complex was prepared according to a previously
described method16 as follows: Na/K tartrate (20.0 mg), Na2CO3 (4.0 mg)
and NaBH4 (7.0 mg) were placed in a vial. The vial was sealed and
flushed with carbon monoxide for 30 min. 99mTc-sodium pertechnetate
(185–1850 MBq, 1 mL) was added and the mixture incubated at 75 ꢀC
for 30 min. After cooling, pH was adjusted to 7.0 with 1 N HCl solution.
The complex formation was checked by HPLC analysis as indicated in
the general experimental section. Retention time: 4.0 min. Radiochemical
purity > 90%.
Conclusions
Labelling of triazole derivative of glucose (5)
A glucose derivative suitable for labelling with 99mTc through the
formation of a Tc(I)-tricarbonyl complex was successfully pre-
pared. The introduction of the histidine-like, 1,4-disubstituted
triazole used as chelator by ‘click chemistry’ and the labelling
by substitution of the precursor were performed in one pot
without further purification. The procedure is fast and simple,
and a single product with adequate radiochemical purity was
obtained. The 99mTc complex was stable in labelling milieu and
exhibited high hidrophilicity and low protein binding. ‘In vivo’
biodistribution profile in mice bearing induced tumours was very
favourable. Although tumour uptake was low, retention up to 2 h
post-injection was considerable, and favourable tumour/muscle
ratios were achieved. Although synthesis and evaluation of
various 99mTc-glucose derivatives have been reported, most of
them have been derivatized in C1 instead of C2, probably because
of easier synthetic conditions.3,4,22 Comparison of biological
results is difficult because of the heterogeneity in animal models
used. No comparison with [18F]-FDG in the same experimental
conditions has been reported to our knowledge. Our data, on
the other hand, indicate that in spite of moderate tumour uptake,
similarity to biodistribution of [18F]-FDG is a promising outcome.
Further modifications of the chelator and linker to increase
lipophilicity might improve biological results.
Neutralized fac-[99mTc(OH2)3(CO)3]+ precursor (50 mL) was added to the
reaction mixture obtained as result of the aforementioned described
deprotection procedure and heated for 1 h at 100 ꢀC. Complex formation
was checked by HPLC analysis. Retention time: 14.0 min. Radiochemical
purity > 90%.
Labelling of propargylglycine and azide derivative of glucose (4)
Neutralized fac-[99mTc(OH2)3(CO)3]+ precursor (50 mL) was mixed with
propargylglicine in water (1.131 mg/mL, 50 mL) and heated for 1 h at
100 ꢀC. Complex formation was checked by HPLC analysis as described
in the general experimental section. Neutralized fac-[99mTc(OH2)3(CO)3]+
precursor (50 mL) was mixed with deprotected azide derivative of glucose
4 and heated for 1 h at 100 ꢀC. Complex formation was checked by HPLC
analysis.
Physicochemical evaluation
Stability in labelling milieu
99mTc complex was incubated in the labelling milieu at room tempera-
ture, and the radiochemical purity was assessed by HPLC for up to 4 h
after labelling.
Protein binding studies
99mTc complexes (25 mL) were incubated with human plasma (475 mL) at
37 ꢀC for up to 120 min. At 30 and 120 min, aliquots (50 mL) were added
to MicroSpin G-50 columns (GE Healthcare Failfield, Connecticut, USA),
which have been pre-spun at 2000 g for 1 min. Columns were centrifuged
again at 2000 g for 2 min, and the collected elute and the column were
counted in a NaI(Tl)-scintillation counter. Protein bound tracer was calcu-
lated as the percentage of activity eluted from the column.
Acknowledgements
This study is partially supported by ANII (Project FCE_140), IAEA
and Pedeciba-Química. We thank Dr M. Moreno for providing cell
lines and Dr R. Alberto and Dr R. Schibli for technical support.
Lipophilicity
Lipophilicity was studied through the apparent partition coefficient
between 1-octanol and phosphate buffer (0.125 M, pH 7.4). In
a
centrifuge tube, containing 2 mL of each phase, 0.1 mL of the 99mTc
complex solution was added, and the mixture was shacked on a Vortex
Conflict of Interest
mixer and finally centrifuged at 5000 r.p.m. for 5 min. Three samples The authors did not report any conflict of interest.
J. Label Compd. Radiopharm 2012, 55 274–280
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

S. Fernández et al.
[12] T. L. Mindt, H. Struthers, L. Brans, T. Anguelov, C. Schweinsberg, V.
Maes, D. Tourwe, R. Schibli, J. Am. Chem. Soc. 2006, 128, 15096.
[13] M. V. Dhyani, D. Satpati, A. Korde, H. D. Sarma, C. Kumar, S. Banerjee,
Nucl. Med. Biol. 2010, 37, 997.
[14] G. Gasser, K. Jäger, M. Zenker, R. Bergmann, J. Steinbach, H. Stephan,
N. Metzler-Nolte, J. Inorg. Biochem. 2010, 104, 1133.
[15] G. Ferro-Flores, I. A. Rivero, C. L. Santos-Cuevas, J. I. Sarmiento, C.
Arteaga de Murphy, B. E. Ocampo-García, R. García-Becerra, D.
Ordaz-Rosado, Appl. Radiat. Isot. 2010, 68, 2274.
References
[1] R. J. Gillies, R. A. Gatenby, Nat. Rev. Cancer 2004, 4, 891.
[2] S. R. Bayly, C. L. Fisher, T. Storr, M. J. Adam, C. Orvig, Bioconjugate
Chem. 2004, 15, 923.
[3] Q.-F. Xiong, Y. Chen, Cancer Biother. Radiopharm. 2008, 23, 376.
[4] M. L. Bowen, C. Orvig, Chem. Commun. 2008, 41, 5077.
[5] L. Branco de Barros, V. Nascimento Cardoso, L. Mota, E. Amaral
Leite, M. de Oliveira, R. J. Alves, Bioorg. Med. Chem. Lett. 2009,
19, 2497.
[16] R. Alberto, R. Schibli, A. Elgi, P. A. Schubiger, J. Am. Chem. Soc. 1998,
[6] L. Branco de Barros, V. Nascimento Cardoso, L. Mota, E. Amaral Leite,
M. de Oliveira, R. J. Alves, Bioorg. Med. Chem. Lett. 2010, 20, 2478.
[7] C. Borges Brasileiro, C. M. da Fonseca Pacheco, C. Martins Queiroz-Junior,
120, 7987.
[17] R. Schibli, C. Dumas, J. Petrig, L. Spadola, L. Scapozza, E. Garcia-Garayoa,
P. A. Schubiger, Bioconjugate Chem. 2005, 16, 105.
C. F. de Lima, J. Batista da Silva, T. Passos Ribeiro de Campos, Appl. [18] E. Pozsgay, E. P. Dubois, L. Panell, J. Org. Chem. 1997, 62, 2832.
Radiat. Isot. 2010, 68, 22.
[19] E. F. V. Scriven, K. Turnbull, Chem. Rev. 1988, 88(2), 297.
[8] C. Decristoforo, I. Santos, H.-J. Pietzsch, A. Duatti, C. J. Smith, A. Rey, [20] E. Benoist, Y. Coulais, M. Almant, J. Kovensky, V. Moreau, D. Lesur, M.
R. Alberto, E. von Guggenberg, R. Haubner, Q. J. Nucl. Med. Mol.
Imaging 2007, 51, 33.
[9] J. Giglio, G. Patsis, I. Pirmettis, M. Papadopoulos, C. Raptopoulou, M.
Pelecanou, E. León, M. González, H. Cerecetto, A. Rey, Eur. J. Med.
Chem. 2008, 43, 741.
[10] J. Giglio, S. Fernández, A. Rey, H. Cerecetto, Bioorg. Med. Chem. Lett.
2011, 21, 394.
Artigau, C. Picard, C. Galaup, S. G. Gouin, Carbohyd. Res. 2011, 346, 26.
[21] J. E. Hodge, C. E. Rist, J. Am. Chem. Soc. 1952, 74, 1498.
[22] R. Alberto, R. Schibli, R. Waibel, U. Abram, A. P. Schubiger, Coord.
Chem. Rev. 1999, 901, 190.
[23] R. Dapueto, R. Castelli, M. Fernández, J. A. Chabalgoity, M. Moreno, J. P.
Gambini, P. Cabral, W. Porcal, Bioorg. Med. Chem. Lett. 2011, 21,
7102.
[11] H. Beckmann, V. Wittmann, Org. Lett. 2007, 9, 1.
[24] X. Chen, L. Li, F. Liu, B. Liu, Bioorg. Med. Chem. Lett. 2006, 16, 5503.
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 274–280