Biological evaluation of glucose and deoxyglucose derivatives radiolabeled with [99mTc(CO)3(H2O)3]+ core as potential melanoma imaging agents

Article abstract of DOI:10.1016/j.bmcl.2011.09.106

Glucose 9 and 2-deoxyglucose 10 were successfully synthesized and radiolabeled with [^99mTc(CO)₃(H₂0) ₃]⁺ intermediate in high yield. The complexes were characterized by HPLC and its stability with his

Full text of DOI:10.1016/j.bmcl.2011.09.106

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7102–7106
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biological evaluation of glucose and deoxyglucose derivatives radiolabeled
with [99mTc(CO)3(H2O)3]+ core as potential melanoma imaging agents
Rosina Dapueto a,b, Romina Castelli b, Marcelo Fernández b, José A. Chabalgoity c,
a,
María Moreno c, Juan Pablo Gambini d, Pablo Cabral c, , Williams Porcal
⇑
⇑
a Laboratorio de Química Orgánica, Igua 4225, Facultad de Ciencias-Facultad de Química, Universidad de la República, 11400 Montevideo, Uruguay
b Laboratorio de Radiofarmacia, Centro de Investigaciones Nucleares, Mataojo 2055, Facultad de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay
c Departamento de Desarrollo Biotecnológico Instituto de Higiene, Av Alfredo Navarro 3051, Facultad de Medicina, Universidad de la Republica, 11600 Montevideo, Uruguay
d Centro de Medicina Nuclear, Hospital de Clínicas, Av Italia s/n, Facultad de Medicina, Universidad de la Republica, 11600 Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
Glucose 9 and 2-deoxyglucose 10 were successfully synthesized and radiolabeled with [99mTc(CO)3(H20)3]+
intermediate in high yield. The complexes were characterized by HPLC and its stability with histidine over
time was challenged. Cell uptake and biodistribution studies in melanoma-bearing C57BL/6 mice were per-
formed. Both compounds showed accumulation in tumor tissue with high tumor-to-muscle ratios. Thus,
Article history:
Received 25 July 2011
Revised 20 September 2011
Accepted 21 September 2011
Available online 1 October 2011
D
-glucose- and
D
-2-deoxyglucose-99mTc complex could be considered as agents for melanoma diagnosis.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Glucose
99m Technetium
Radiopharmaceutical
Melanoma
Tumor diagnostic
Glucose is the major energy source of cells, especially in heart
and brain. Cancer cells are well known to display an enhanced car-
bohydrate uptake and consumption.1,2 Due to the ideal decay prop-
While some of these biomolecules showed their potential in preli-
minary evaluations, only few of these agents achieved clinical sig-
nificance. Therefore, our goal is to develop diagnostic imaging
agents with high specificity and sensitivity towards melanoma,
using B16F1 model for in vivo and in vitro experimentation. In this
context, we propose the evaluation of 99mTc-glucose derivatives.
Here, we report the potential use of two 99mTc labeled-glucose
derivatives for melanoma diagnosis by evaluation in B16F1 mela-
noma-bearing C57BL/6 mice and cell uptake in B16F1 murine mel-
anoma cells.
It is of great importance to achieve the effective complexation
of the metal core with the biological active molecule without
affecting its physiological properties. In the last years, the organo-
metallic aqua complex [99mTc(H2O)3(CO)3]+ has been proposed as a
versatile molecule to form the tricarbonyl core [99mTc(CO)3]+ moi-
ety.10,23 Tricarbonyl technetium-99m is an attractive core for the
introduction of 99mTc into biomolecules because of its high chem-
ical stability and small size. This precursor can easily form com-
plexes with different di- or triligands by substitution of two or
three water molecules, respectively. For the purposes of labeling
glucose and 2-deoxyglucose with 99mTc, a hydrophilic iminodiace-
tic acid (IDA) chelate was attached to position C-1 separated by an
ethylene linker (Scheme 1). Coordination of the tricarbonyl core is
likely to be tridentate, via the two carboxylate oxygens and the ter-
tiary amine of the IDA moiety.
erties (t1/2 = 6 h,
c energy = 150 keV), low cost of production and
on-site availability, 99mTc is the most frequently used radioisotope
in nuclear medicine today.9,10 Several groups have reported the
preparation of glucose analogues labelled with 99mTc which dem-
onstrate uptake into tumor tissue indicating that they are potential
agents for use in metabolic imaging.11–13 In addition, we recently
described the use of 99mTc-glucarate as a potential agent for breast
cancer imaging.14
Our group has recently started to develop potential radiophar-
maceuticals for imaging melanoma. Melanoma is known as one of
the most aggressive tumors in humans.15,16 Its incidence has been
steadily increasing over the past years, making it a serious problem
for healthcare in several countries worldwide. Therefore, an early
diagnosis of this disease together with an accurate assessment of
its metastases could be the key for disease survival.17,18 Several
scintigraphic studies using monoclonal antibodies,19 peptides,20
iodobenzamides21 and carbohydrates22 have been developed over
the past years to detect melanoma and localize metastatic lesions.
⇑
Corresponding authors. Tel./fax: +598 2 525 0800 (P.C.); tel.: +598 2 525 8618;
fax: +598 2 525 0749 (W.P.).
E-mail addresses: pcabral@cin.edu.uy (P. Cabral), wporcal@fq.edu.uy (W. Porcal).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.106

R. Dapueto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7102–7106
7103
OAc
O
OAc
AcO
AcO
a
O
OAc
1
Br
c, d
AcO
AcO
O
OH
OAc
2
R
b
3 R=OAc (β)
O
4
α
R = H ( )
AcO
AcO
2
OEt
OAc
OAc
O
O
O
e, f
AcO
AcO
AcO
AcO
O
N
O
N3
O
2
2
R
R
5 R=OAc (β)
7 R=OAc (β)
8 R = H (α)
6
α
R = H ( )
OEt
O
OH
ONa
O
g
HO
O
N
HO
2
R
9 R=OH (β)
10 R = H (α)
O
ONa
Scheme 1. Synthesis of glucose and 2-deoxyglucose derivatives. Reagents and conditions: (a) Diethylene glycol, HgBr2, molecular sieves, CH2Cl2, 71%; (b) Diethylene glycol,
acid resin (WPX-800), molecular sieves, acetonitrile, 40%; (c) TsCl, pyridine, CH2Cl2, 3 72% 4 60%; (d) NaN3, DMF, 60 °C, 3 h, 5 92% 6 80%; (e) (i) Triphenylphosphine polymer-
bound, CH2Cl2, (ii) H2O; (f) Ethyl bromoacetate, Et3N, THF, 7 21% 8 24%; over 2 steps; (g) (i) NaOMe, MeOH, (ii) NaOH, H2O, quantitative yields.
Synthesis of 99mTc-glucose derivatives was performed using the
method previously described with some modifications (Schemes 1
and 2).24 Glycosylation of commercially available acetobromoglu-
further purification.26 Subsequently, the IDA moiety was built up in
one step by a double alkylation of the corresponding amine with
ethyl bromoacetate to yield 7 and 8.26 Finally, the desired disodium
salts 9 and 10, were obtained in quantitative yields by deprotection
of the acetates in two steps.24 First the acetates were removed with
sodium methylate and then the esters were hydrolyzed with 1 M
NaOH.
cose 1 and tri-O-acetyl-D-glucal 2 with ethylene glycol resulted
in alcohols 3 and 4, in good and moderate yield, respectively. The
esters 7 and 8 were obtained through reduction-N-alkylation of
the corresponding azides. To obtain the amines intermediates a
Staudinger reaction has been adapted using a triphenylphosphine
polymer-bound (TPP-PB).25 The azide 5 and 6 were dissolved in
dry THF and TPP-PB was added. After 24 h at room temperature,
water was added and the reaction was stirred at room temperature
for 6 h. Following standard work-up protocols and analysis, the de-
sired amines were obtained and used in the next reaction without
For radiolabelling, precursor
[
99mTc(H2O)3(CO)3]+ was pre-
pared27 by boranocarbonate reduction from a saline solution of
Na99mTcOꢀ4 , using an IsoLink™ kit generously donated by Mal-
linckdrot Inc. (Scheme 2). Radioactive labeling of 9 and 10 was
accomplished when [99mTc(H2O)3(CO)3]+ was added and the mix-
tures heated at 70 °C for 30 min, as described by Petrig et al.24
O
OH
CO
O
Tc
OC
OC
H2O
H2O
a
CO
CO
b
O
-
99mTcO4
Tc
H2O
HO
HO
O
N
2
R
CO
O
[
[
99mTc]9 R=OH (β)
99mTc]10 R = H (α)
O
Scheme 2. Radiolabeled of glucose 9 and 2-deoxyglucose 10 analogues with [99mTc(CO)3(H2O)3]+ intermediate. Reagents and conditions: (a) IsoLink kit, 100 °C, 30 min; (b) 9
or 10, 70 °C, 30 min, PBS buffer pH 7.4.

7104
R. Dapueto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7102–7106
Table 1
The products were analyzed for their radiochemical purity by
HPLC.28 Radiochemical yield of 99mTc]9 and 99mTc]10 was
99.40% and 99.90%, respectively, immediately after the prepara-
tion. The specific radioactivity of the products was calculated to
Percentage of [99mTc]9 and [99mTc]10 remaining after incubation at 37 °C in 1 mM
[
[
histidine for 1, 4 and 24 h
1 h
4 h
24 h
99mTc]9
99.5 0.2
99.2 0.7
99.2 0.2
97.8 0.9
94.1 1.4
95.7 1.6
be 19.04ꢀ204.0 MBq/
l
mol and 18.5ꢀ77.62 MBq/
lmol, respec-
[
[
99mTc]10
tively. HPLC analysis of the reaction mixtures showed the forma-
tion of main radiochemical species, at 8.3 and 8.9 min,
corresponding to complexes [99mTc]9 and [99mTc]10, respectively
(Fig. 1), while that of [99mTc(CO)3(H2O)3]+ was found to be
3.8 min (result not shown).
Table 2
Biodistribution pattern of 99mTc-glucose complexes [99mTc]9 and [99mTc]10 in normal
C57BL/6 mice, n = 3, at 60 min post-injection
To assess the stability of the complexes, challenge studies were
done with histidine. The radiochemical purity was measured at dif-
ferent time points during the incubation with the amino acid, a po-
tential competitor of metal binding in vivo (Table 1). [99mTc]9 and
% ID/g
60 min Post-injection
Organ
[
99mTc]9
[
99mTc]10
[
99mTc]10 were added to a solution of histidine and incubated at
Blood
Liver
Heart
Lung
Spleen
Kidney
Thyroid
Muscle
Bone
1.65 0.07
7.2 0.8
1.2 0.35
7.7 1.3
37 °C for 24 h, as described by Ferreira et al.29 HPLC samples were
analyzed at 1, 4 and 24 h of incubation of each complex. The tri-
dentate ligand was expected to form a stable complex with the me-
tal core, resistant to ligand exchange process. Only insignificant
decomposition of the complexes occured over 24 h of incubation
with 100-fold excess of histidine, suggesting a high in vivo stabil-
ity. Percentage of the remaining [99mTc]9 and [99mTc]10 were 94.1%
and 95.7%, respectively, shown by HPLC analysis after 24 h incuba-
tion (Table 1).
0.85 0.21
1.3 0.4
0.42 0.24
4.3 1.7
0.28 0.08
1.1 0.21
0.31 0.18
1.8 0.3
0.48 0.1
0.14 0.02
0.16 0.04
0.19 0.05
0.31 0.13
0.14 0.05
0.16 0.07
0.04 0.01
Brain
% of injected dose (% ID)
Partition coefficients were determined between 1-octanol and
PBS at pH 7.4.30 ꢀ1.60 0.11 and ꢀ1.09 0.02 values were found
for [99mTc]9 and [99mTc]10, respectively.
Intestine
Urine and bladder
16 4.3
56 20
35 6.6
42 8.2
Biodistribution studies were carried out on C57BL/6 normal
mice and C57BL/6 mice with B16F1 murine melanoma tumor, at
1.0 h post-injection, following procedure our group recently de-
scribed.31 Animal studies were carried out in compliance with
the national laws related to the ethics during animal experimenta-
tion. B16F1 melanoma cells were inoculated subcutaneously in
studies in melanoma-bearing C57BL/6 mice showed similar pat-
tern to normal mice (Table 3). The complexes demonstrated tumor
accumulation, reaching tumor to muscle ratios of 2.5 0.7 and
2.0 0.3 at 1 h post-injection for [99mTc]9 and [99mTc]10, respec-
tively (Table 4). Similar biodistribution patterns between these
two complexes were observed.
C57BL/6 mice and were used 10 days after.
[
99mTc]9 and
99mTc]10 were injected intravenously and the injected radioactiv-
Tumor-cell uptake of [99mTc]9 and [99mTc]10 was preformed as
[
described by Castelli et al.31 B16F1 murine melanoma cells cul-
tured in D-glucose free media were used in order to determinate
ity was measured with a NaI(Tl) detector. Mice were sacrificed at
1 h post-injection. Tumor, other organs of interest and blood were
collected, weighed and measured for radioactivity. The results are
expressed as the percentage uptake of injected dose per gram of
tissue (%ID/g). Tumor to muscle and tumor to blood ratios were
calculated from the corresponding tissue concentrations. All data
is presented as mean average standard deviation.
whether the complexes are transported intracellularly via GLUT’s.
Figure 2 shows the result of this assay. Internalization of glucose
9 and deoxyglucose 10 labeled with 99mTc in melanoma cells was
demonstrated by measuring the activity remaining within the cells
at different time points. The results suggested that [99mTc]9 and
Biodistribution pattern for [99mTc]9 and [99mTc]10 in C57BL/6
normal mice is shown in Table 2. The complexes were rapidly elim-
inated through kidneys within an hour since more than 50% of the
radioactivity was measured in urine and bladder: 56 20% DI for
[
99mTc]10 are transported into cells. The highest internalizations
reached 18% and 52% of the total activity after 90 min of incubation
with [99mTc]9 and 30 min with [99mTc]10, respectively. On the
other hand, a blocking experiment using
D-glucose was carried
[
99mTc]9 and 42 8.2% DI for [99mTc]10. These results are in agree-
out in order to determinate whether the cellular uptake is GLUT’s
ment with the hydrophilic logP values found, being [99mTc]9 the
highest hydrophilic one. Complexes also showed liver
(7.2 0.8% DI/g for [99mTc]9 and 7.7 1.3% DI/g for [99mTc]10) and
specific. Under these conditions, only [99mTc]9 complex uptake
was blocked (50% blockage) in presence of D-glucose, suggesting
that internalization could be via GLUT’s transporters.
In summary, labeling of glucose 9 and 2-deoxyglucose 10 via
the 99mTc-tricarbonyl precursor was achieved in high yields
(>99%). In our proposed design, the addition of a chelating moiety
as IDA to the carbohydrate could minimize the perturbation of
intestine (16 4.3% DI for
[
99mTc]9 and 35 6.6% DI for
[
99mTc]10) excretion, since activity accumulated in excretory or-
gans within an hour and were rapidly cleared from blood and mus-
cle. Uptake in other organs was insignificant. Biodistribution
Figure 1. HPLC chromatographys obtain for [99mTc]9 (left) and [99mTc]10 (right) after radiolabeling procedure with [99mTc(CO)3(H2O)3]+ of 9 and 10. (Solvents: A = Water/TFA
0.1%, B = Acetonitrile/TFA 0.1%, Gradient: 100% A, 0% B time = 0 min, 0% A, 100% B t = 20 min, 100% A, 0% B t = 22 min, flow rate = 1 mL/min). CPS: counts per second.

R. Dapueto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7102–7106
7105
Table 3
tribution studies showed that both complexes behave similar and
have almost identical tumor to muscle and tumor to blood ratios
meaning that deoxyglucose have the same distribution in vivo than
glucose. The results presented here justify further investigations in
animals and humans of these complexes as potential melanoma
imaging agents.
Biodistribution pattern of 99mTc-glucose complexes [99mTc]9 and [99mTc]10 in B16F1
melanoma-bearing C57BL/6 mice, n = 3, at 60 min post-injection
% ID/g
60 min Post-injection
Organ
[
99mTc]9
[
99mTc]10
Blood
Liver
Heart
Lung
Spleen
Kidney
Thyroid
Muscle
Bone
1.6 1.3
3.9 0.99
0.31 0.14
0.67 0.49
0.27 0.11
2.3 0.95
0.39 0.14
0.12 0.02
0.17 0.04
2.3 0.28
8.7 1.3
0.38 0.06
1.5 0.57
0.38 0.22
3.5 0.71
0.41 0.16
0.29 0.08
0.46 0.30
0.15 0.02
0.40 0.28
Acknowledgments
This work was supported by CHLCC-Uruguay and CSIC-UdelaR.
We thank CHLCC-Uruguay and CSIC-UdelaR for scholarship to R.D.
References and notes
Brain
Tumor
0.046 0.006
0.31 0.23
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Rev. Dermatol. 2008, 3, 569.
Intestine
Urine and bladder
19 4.1
65 7.6
46 6.2
16 2.1
Table 4
Tumor/blood and tumor/muscle ratios for 99mTc-glucose complexes ([99mTc]9 and
99mTc]10) at 60 min post-injection
[
16. Oliveria, S. A.; Saraiya, M.; Geller, A. C.; Heneghan, M. K.; Jorgensen, C. Arch. Dis.
Child. 2006, 91, 131.
Tumor/blood
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18. Belhocine, T. Z.; Scott, A. M.; Even-Sapir, E.; Urbain, J. L.; Essner, R. J. Nucl. Med.
2006, 47, 957.
[
[
99mTc]9
0.21 0.05
0.24 0.02
2.5 0.3
2.0 0.7
99mTc]10
19. Camacho, X.; García, M. F.; Balter, H.; Fernández, M.; Porcal, W.; Gambini, J. P.;
Alonso, O.; Cabral, P. Nucl. Med. Biol. 2010, 37, 697.
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2009, 20, 583.
22. Yamada, K.; Brink, I.; Bissé, E.; Epting, T.; Engelhardt, R. J. Dermatol. 2005, 32,
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[
[
99m Tc] 9
99m Tc] 10
100000
80000
60000
40000
20000
0
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1868.
25. Lindsley, C. W.; Zhao, Z.; Newton, R. C.; Leister, W. H.; Strauss, K. A. Tetrahedron
Lett. 2002, 43, 4467.
26. General procedure for preparation of esters 7 and 8: Azide 5 or 6 (1 equiv) was
dissolved in dry CH2Cl2 (24 mL/mmol) and triphenylphosphine polymer-bound
(Sigma, 2 equiv, 3 mmol/g) was added. After the mixture had been shaked for
24 h at room temperature, water (0.38 mL/mmol) was added and then was
shaked for a further 6 h. The mixture was filtered, washed with CH2Cl2 and the
solvent was evaporated in vacuo to obtain the corresponding amines, that were
sufficiently pure by TLC analysis for further synthesis. Immediately,
triethylamine (2.2 equiv) and ethyl bromoacetate (2.2 equiv) were added to a
solution of the corresponding amines in dry THF (17 mL/mmol). The solution
was refluxed for 5 h and then was stirred overnight at room temperature under
15
30
60
90
N2 atmosphere. The mixture was filtered, diluted with CH2Cl2 and washed with
90 (D-Glucose)
water. The organic phases were dried over Na2SO4 and the solvent was
evaporated in vacuo. The crude was subjected to column cromatography (SiO2,
mixtures of Hexane/Ethyl acetate 3:7) to afford 7 and 8. (7): 23%, yellowish oil,
1H-RMN (CDCl3): d = 5.21 (t, J = 9.2 Hz, 1H; H-3), 5.09 (t, J = 9.6 Hz, 1H; H-4),
5.00 (t, J = 8.0 Hz, 1H; H-2), 4.63 (d, J = 8.0 Hz, 1H; H-1), 4.29 (dd, J = 4.8 Hz,
J0 = 4.0 Hz, 1H; H-6a), 4.18 (c, J = 7.2 Hz, 4H; OCH2CH3), 4.12 (m, 1H; H-6b), 3.99
(m, 1H; OCH2CH2N), 3.71 (m, 2H; H-5 OCH2CH2N), 3.57 (s, 4H; NCH2C@O), 3.04
(m, 2H; OCH2CH2N), 2.08, 2.04, 2.02, 1.99 (s, 12H; CH3C@O), 1.28 (t, J = 7.2 Hz,
6H; OCH2CH3). 13C-RMN (CDCl3): d = 7.5 (OCH2CH3), 53.5 (OCH2CH2N), 55.2 (C-
5), 56.3 (NCH2C@O), 61.6 (OCH2CH3), 62.5 (C-6), 68 (C-4), 69.2 (OCH2CH2N),
71.7 (C-2), 73.1 (C-3), 101.5 (C-1), 170.3 ((C@O)Me), 171.6 ((C@O)Et). (8): 37%,
yellowish oil, 1H-RMN (CDCl3): d = 5.32 (m, 1H; H-3), 5.04 (m, 2H; H-1 H-4),
4.36 (dd, J = 4.4 Hz J0 = 4.4 Hz, 1H; H-6a), 4.22 (c, J = 6.8 Hz, 4H; OCH2CH3), 4.07
(d, J = 2.0 Hz, 1H; H-6b), 4.04 (m, 1H; H-5), 3.80 (m, 1H; OCH2CH2N), 3.63 (s,
4H; NCH2C@O), 3.61 (m, 1H; OCH2CH2N), 3.05 (m, 2H; OCH2CH2N), 2.26 (dd,
J = 0.8 Hz J0 = 1.2 Hz, 010 H; H-2 ec.), 2.11, 2.06, 2.02 (s, 9H; CH3C@O), 1.87 (ddd,
Time (min)
Figure 2. Tumor-cell uptake assay of 99mTc-glucose complexes ([99mTc]9 and
99mTc]10) by B16F1 cells at 37 °C during 15, 30, 60 and 90 min. Blocking
experiment was performed by incubation with -glucose for 90 min. Total
radioactivity is presented as count per minute (CPM).
[
D
structure and function. Other chelating systems as MAG3 or
MAMA, are also suitable options for radiolabeling glucose with
technetium-99m as describe by Chen et al.13 Those complexes pre-
sented similar biodistribution patterns, tumor uptake and partition
coefficients than the complexes described in this Letter. [99mTc]9
and [99mTc]10 complexes were evaluated for its cell internaliza-
tion and biodistribution in C57BL/6 mice bearing B16F1 murine
melanoma model. The high in vitro uptake of [99mTc]10 which
was not dependent of glucose, supporting the idea that different
affinity glucose transporters isoforms or passive transport may
contribute to glucose transport across the cell membrane. Biodis-
´
J = 3.6 Hz J = 7.8 Hz J = 3.6 Hz, 1H; H-2ax), 1.31 (t, J = 6.8 Hz, 6H; OCH2CH3).
13C-RMN (CDCl3): d = 14.1 (OCH2CH3), 35.5 (C-2), 54.5 (OCH2CH2N), 56.7
(NCH2C@O), 60.3 (OCH2CH3), 62.5 (C-6), 67.2 (C-5), 67.7 (OCH2CH2N), 69.7 (C-
4), 98.0 (C-1), 170.1 ((C@O)Me), 171.5 ((C@O)Et).
27. Na½99mTcO4ꢀꢁ (1 mL, 200 MBq) from 99mTc/99Mo generator was added to
Isolink™ kit. The solution was incubated in water bath at 100 °C for 20 min
and then was neutralized with 1 mL of 0.1 N HCl solution. Label was controlled
by HPLC as described in this Letter.

7106
R. Dapueto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7102–7106
28. HPLC: Agilent 1200 system control equipped with gamma detector Raytest,
30. Decristoforo, C.; Faintuch-Linkowski, B.; Rey, A.; von Guggenberg, E.; Rupprich,
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