99mTc(I)/Re(I) tricarbonyl complexes for in vivo targeting of melanotic melanoma: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.02.014

The ^99mTc (I) tricarbonyl complexes fac-[ ^99mTc(κ³-L)(CO)₃] (Tc1-Tc6) containing N-ethylpyrrolidine and N,N-diethylethylamine groups for melanin binding, were evaluated in vitro and in vivo as radioactive probes for the targeting of melanotic melanoma. Aiming at the modification of their size, topology and lipophilicity, Tc1-Tc6 were obtained based on an S,N,O-donor bifunctional chelator (BFC) derived from cysteamine and on pyridyl- and pyrazolyl-containing N,N,O-donor BFCs. Tc1-Tc6 were chemically identified by HPLC comparison with the Re congeners (Re1-Re6) that were synthesized at the macroscopic level and fully characterized by common analytical techniques. With the exception of Tc5 and Tc6, these ^99mTc complexes are moderately lipophilic, and bind to melanin with moderate to high affinity (23-87%). The cell uptake of Tc1-Tc6, expressed as a percentage of total activity per million cells, spanned between 0.86 and 21.02% for the melanotic B16-F1 cell line and between 0.49% and 13.58% for the amelanotic A375 cell line. In the B16-F1 cell line, Tc1, Tc3 and Tc4 showed moderate cellular uptake values (>10% at 4 h of incubation). In the amelanotic A375 cell line, only Tc4 has shown a moderate cell uptake (>10% at 4 h of incubation), with all the other compounds displaying a relatively poor uptake, i.e. inferior to 5%. Competition studies with haloperidol have shown that the involvement of sigma receptors in cellular uptake and retention is likely to occur for Tc4. Complex Tc1, stabilized with the S,N,O-donor BFC and containing a N,N-diethylethylamine group, presented the most promising biological profile for in vivo targeting of melanoma, showing a moderate tumor uptake of 2.17% ID/g at 1 h p.i in a B16-F1 melanoma-bearing mouse and rather favorable target/non-target ratios with values as high as 16.9 and 5.2 for tumor/muscle and tumor/blood ratios, respectively.

Full text of DOI:10.1016/j.ejmech.2012.02.014

European Journal of Medicinal Chemistry 50 (2012) 350e360
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
99mTc(I)/Re(I) tricarbonyl complexes for in vivo targeting of melanotic melanoma:
Synthesis and biological evaluation
Carolina Moura a, Lurdes Gano a, Filipa Mendes a, Paula D. Raposinho a, A.M. Abrantes b, M.F. Botelho b,
,
Isabel Santos a, António Paulo a
*
a Unidade de Ciências Químicas e Radiofarmacêuticas, ITN, Estrada Nacional 10, 2686-953 Sacavém, Portugal
b Biophysics/Biomatematics Institute, CIMAGO, IBILI, Faculty of Medicine, University of Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
The 99mTc (I) tricarbonyl complexes fac-[99mTc( 3-L)(CO)3] (Tc1eTc6) containing N-ethylpyrrolidine and
k
Article history:
Received 7 December 2011
Received in revised form
3 February 2012
Accepted 6 February 2012
Available online 14 February 2012
N,N-diethylethylamine groups for melanin binding, were evaluated in vitro and in vivo as radioactive
probes for the targeting of melanotic melanoma. Aiming at the modification of their size, topology and
lipophilicity, Tc1eTc6 were obtained based on an S,N,O-donor bifunctional chelator (BFC) derived from
cysteamine and on pyridyl- and pyrazolyl-containing N,N,O-donor BFCs. Tc1eTc6 were chemically
identified by HPLC comparison with the Re congeners (Re1eRe6) that were synthesized at the macro-
scopic level and fully characterized by common analytical techniques. With the exception of Tc5 and Tc6,
these 99mTc complexes are moderately lipophilic, and bind to melanin with moderate to high affinity (23
e87%). The cell uptake of Tc1eTc6, expressed as a percentage of total activity per million cells, spanned
between 0.86 and 21.02% for the melanotic B16-F1 cell line and between 0.49% and 13.58% for the
amelanotic A375 cell line. In the B16-F1 cell line, Tc1, Tc3 and Tc4 showed moderate cellular uptake
values (>10% at 4 h of incubation). In the amelanotic A375 cell line, only Tc4 has shown a moderate cell
uptake (>10% at 4 h of incubation), with all the other compounds displaying a relatively poor uptake, i.e.
inferior to 5%. Competition studies with haloperidol have shown that the involvement of sigma receptors
in cellular uptake and retention is likely to occur for Tc4. Complex Tc1, stabilized with the S,N,O-donor
BFC and containing a N,N-diethylethylamine group, presented the most promising biological profile for
in vivo targeting of melanoma, showing a moderate tumor uptake of 2.17% ID/g at 1 h p.i in a B16-F1
melanoma-bearing mouse and rather favorable target/non-target ratios with values as high as 16.9 and
5.2 for tumor/muscle and tumor/blood ratios, respectively.
Keywords:
Technetium-99m
Rhenium
Radiopharmaceuticals
Melanin
Melanoma
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
At present, 2-[18F]fluoro-2-deoxy- -glucose (18F-FDG) is the
D
unique radioactive probe in clinical use for the detection of mela-
noma. However, 18F-FDG is a nonspecific PET tumor imaging agent
with serious limitations for the diagnostic of melanoma due to its
poor sensitivity to detect micrometastic sites [3,4]. For this reason,
alternative target-specific probes for the diagnostic of melanoma,
by means of PET or SPECT, have been investigated in the past few
years. In this area of research, particular effort has been devoted to
the design of compounds having the ability to interact with intra-
cellular melanin, which is a very attractive target for melanoma
diagnosis due to the extensive pigmentation of most melanoma
cells [5]. A considerable part of this research work involved 123I-
labeled benzamide derivatives, which led in some instances to
encouraging results justifying their clinical evaluation as SPECT
probes for in vivo detection of melanoma [6e13]. Radioiodinated
benzamides seem to be localized in the melanocytes of pigmented
cells and their uptake has been correlated to melanin binding,
Malignant melanoma is one of the most lethal cancers due to its
high cellular proliferation rate and the early occurrence of metas-
tases. Around 160,000 new cases of melanoma are diagnosed in the
world each year, and although procedures for effective treatment of
melanoma are still not available, increased surveillance with early
diagnosis and accurate staging of the disease is an important
approach to increase survival [1,2]. The nuclear imaging techniques
Positron Emission Tomography (PET) and Single Photon Emission
Computed Tomography (SPECT) may allow such early diagnosis,
provided that radioactive probes are available for in vivo targeting
of melanoma and its metastases.
* Corresponding author.
E-mail address: apaulo@itn.pt (A. Paulo).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.014

C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
351
suggesting a non-receptor uptake mechanism that, however, is not
fully understood [10,14e18].
liphophilicity, anchored by a new linear (N,S,O)-donor ligand
framework derived from cysteamine [29]. The preliminary in vitro
evaluation of Tc1 has shown that this complex displays a moderate
cell uptake in B16-F1 murine melanoma cells. These encouraging
findings prompted us to pursue with the biological evaluation of
Tc1, and to extend our studies to congener neutral complexes of
pyridyl- and pyrazolyl-containing (N,N,O)-donor bifunctional
chelators [30,31] functionalized with N,N-diethylamine and N-
pyrrolidine groups for melanin binding (Scheme 1). By exploring
different classes of chelators, we expected to have a better ability to
tune the physico-chemical properties of the final complexes, such
as size, topology and lipophilicity, which can be determinant of
their ability to freely diffuse across the cell membrane and to
interact with the putative cytosolic target. In these complexes, the
metallic chelate containing the fac-[99mTc(CO)3]þ unit would
replace the aromatic part of benzamides while the pendant and
protonable N,N-dialkylalkyl group should enhance their affinity
towards the melanin pigment.
123I-labeled molecules present several disadvantages for clinical
diagnosis by SPECT compared with 99mTc-based radiopharmaceuti-
cals, since 99mTc has more favorable nuclear properties for routine
clinical imaging and is available at lower costs from a 99Mo-99mTc
generator [19]. Therefore, several research groups embarked in the
search of 99mTc complexes for the in vivo detection of melanoma in
alternative to radioiodinated benzamides. This involved mainly the
synthesis of 99mTc complexes functionalized with benzamide
derivatives or their fragments, with the aim of obtaining compounds
still behaving as melanin binders and with the ability to reach this
pigment at intracellular level. For this purpose, a variety of
complexes have been studied, including Tc(V) complexes with the
[
99mTcN]2þ [20e22] and [99mTcO]3þ [19,23e25] cores stabilized by
N2S2 tetradentate BFC’s, [3 þ 1] mixed ligand Tc(V) oxocomplexes
[26,27] and, more recently, organometallic complexes with the fac-
[
99mTc(CO)3]þ [12,28] core. None of these complexes has emerged as
a good alternative to radioiodinated benzamides, despite some
encouraging results found for Tc(V) oxocomplexes bearing the N,N-
diethylamine group as a melanin-binding pharmacophore.
Herein, we report on the synthesis and characterization of new
Re (Re2eRe6) and 99mTc (Tc2eTc6) tricarbonyl complexes,
anchored by (S,N,O)- or (N,N,O)-donor BFCs and bearing N,N-
diethylamine and N-pyrrolidine groups that have been linked at the
central amine of the chelators using an ethylenic linker (Scheme 1).
We also compare the biological behavior of Tc2eTc6 with the one
exhibited by the previously reported Tc1, aiming to have an insight
into their relevance for the design of 99mTc-labeled probes for
in vivo targeting of melanotic melanoma. To obtain such insight, the
biological evaluation of these 99mTc complexes comprised the
measurement of their binding affinity to synthetic melanin, cellular
uptake studies in the melanotic murine B16-F1 cell line and the
amelanotic A375 human cell line, as well as biodistribution studies
in melanoma-bearing C57BL/6 female mice. In addition, competi-
Looking for compounds with interest for the design of SPECT
probes for the in vivo detection of melanotic melanoma, our
research group has described recently Re(I) and Tc(I) complexes
stabilized by pyrazole-diamine and pyrazole-aminocarboxylic
chelators, acting respectively as (N,N,N) and (N,N,O) donors, and
containing the 4-amino-N-(2-diethylaminoethyl)benzamide group
or its fragments [28]. Most of these complexes have shown
a hydrophilic character and high in vitro affinity for melanin, but
presented a low in vitro cell uptake in murine melanoma cells, as
well as a negligible tumor uptake in melanoma-bearing mice.
Searching for complexes with a better ability to target intracellular
melanin in melanotic melanoma cells, we have focused on related
Re(I) and 99mTc(I) tricarbonyl complexes (Re1 and Tc1, respectively)
(Scheme 1) of low molecular weight and with augmented
tion studies with haloperidol, a nonselective s1es2 inhibitor, were
also performed to ascertain the possible influence of sigma recep-
tors in the cell uptake of the complexes.
Scheme 1. Molecular structures of the Re and 99mTc complexes containing N,N-diethylethylamine and N-ethylpyrrolidine groups for melanin binding.

352
C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
2. Results and discussion
to moderate yield (25%e71%). These Re complexes were applied as
reference compounds to assign the chemical identity of the 99mTc
congeners by means of HPLC comparison, a common and well
accepted practice in radiopharmaceutical chemistry due to the
physico-chemical similarities of these group 7 metals.
2.1. Synthesis and characterization of the bifunctional chelators and
respective Re(I) and 99mTc(I) tricarbonyl complexes
The functionalization of the three classes of (S,N,O)- or (N,N,O)-
donor BFCs with the two different N,N-dialkylalkyl groups, N,N-
diethylethylamine and N-ethylpyrrolidine, has been done in
a convergent way using a common synthetic approach. As shown in
Scheme 2, this involved the N-alkylation of the central amine of
each chelator with an appropriate chloro derivative of the N-dia-
lkylalkyl groups. These N-alkylation reactions were performed
under reflux in a THF/H2O mixture at pH 12, by reacting 2-[2-(3,5-
dimethyl-1H-pyrazol-1-yl)ethylamino]acetic acid, 2-[(pyridin-2-
yl)methylamino]acetic acid or 2-[2-(ethylthio)ethylamino]acetic
acid with 1-(2-chloroethyl)pyrrolidine or 2-chloro-N,N-dieth-
ylethanamine (Scheme 2). The resulting new ligands, L2HeL6H,
were obtained in low to moderate yield (15e36%) after adequate
work-up.
All the new compounds, L2HeL6H and Re2eRe6, were charac-
terized by the common spectroscopic techniques (IR, 1H and 13C
NMR), by ESI-MS and elemental analysis. The collected analytical
data confirmed the proposed formulations. In particular, the IR
spectra of Re2eRe6 showed the presence of two strong n(C^O)
bands characteristic of the fac-[Re(CO)3]þ moiety, with values
ranging from 1866 to 2037 cmꢀ1 and comparable to those previ-
ously reported for other Re(I) tricarbonyl complexes anchored by
the same BFCs [30,32]. The 1H NMR spectra of Re3eRe6 showed the
presence of a set of well-defined multiplets in the aliphatic region,
between 2.59 and 4.80 ppm, presenting a splitting pattern and
relative intensities consistent with the diastereotopic character of
the methylenic protons from the coordinating backbone of the
respective (N,N,O)-donor chelators. In these spectra, the aromatic
resonances from the pyridyl and pyrazolyl rings range between
7.53-8.83 ppm and 5.83e5.84 ppm, respectively, being downfield
shifted compared to the same resonances in the corresponding free
ligands. Altogether, these data corroborate the tridentate and facial
The new chelators, L2HeL6H, were reacted with fac-
[Re(H2O)3(CO)3)]Br in refluxing methanol, affording the Re(I) tri-
carbonyl complexes Re2eRe7 (Scheme 3). Re2eRe7 were purified
by column chromatography or by RP-HPLC, being recovered in low
Scheme 2. Synthesis of the ligands.

C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
353
Scheme 3. Synthesis of the Re and 99mTc complexes.
coordination of L2eL6. For complex Re2, the 1H and 13C NMR
spectra recorded at room temperature indicated the presence of
two interconverting species, due to sulfur inversion as we have
reported for the similar compound, Re1, containing the same S,N,O-
donor atom set but bearing a N,N-diethylamine substituent. The 1H
NMR spectra of Re2 were run at variable temperature, from ꢀ40 ꢁC
to 60 ꢁC , but the effect of the temperature on the resonances of
each invertomer could not be followed properly due to the
complexity of the spectra. The 13C NMR spectrum obtained at 20 ꢁC
presented two relatively narrow resonances for each methylenic
carbon in agreement with the presence of two interconverting
invertomers. By raising the temperature to 60 ꢁC, each pair of these
resonances merged to single signals, which are relatively narrow
for the CH2 atoms of the linker and N-pyrrolidine ring but are rather
broad for the methylenic carbons linked to the coordinating S, N,
and O atoms. These data show that a fast-exchange limit spectrum
could not be obtained at this temperature, indicating also that the
sulfur inversion affects more strongly the chemical environment of
the atoms that are closer to the metallic center.
The 99mTc complexes, Tc2eTc6, were synthesized in aqueous
solution at pH 5.5 by reaction of fac-[99mTc(H2O)3(CO)3]þ with the
appropriate ligand (L2eL6) at 100 ꢁC for 30 min, as previously re-
ported for Tc1 [29]. Under these conditions, all complexes were
obtained with a high radiochemical yield (ꢂ95%) being used in the
in vitro and in vivo biological studies without further purification.
The chemical identification of Tc2eTc6 has been done by compar-
ison of their HPLC profiles with those of the corresponding rhenium
complexes (Re2eRe6). The in vitro evaluation of Tc2eTc6 involved
the measurement of their lipophilicity and the evaluation of their
binding to synthetic melanin (Table 1).
The lipophilicity, expressed as the distribution coefficient (log
Do/w) in octanol/0.1 M phosphate buffer (pH 7.4), was determined
using the multiple back-extraction method [33]. With the excep-
tion of Tc5 and Tc6, displaying log Do/w values of 0,00 ꢃ 0,01
and ꢀ0,02 ꢃ 0,03 respectively, all the other complexes are lipo-
philic with log Do/w values ranging between 0.87 and 1.12.
The measurement of the in vitro affinity of Tc2eTc6 to melanin
involved the incubation of the complexes at room temperature for

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C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
Table 1
HPLC retention times, log D values and in vitro binding to synthetic melanin for
complexes Tc1eTc6.
Compound
Retention
log Do/w (pH 7.4)
% Bound to
synthetic melanin
time (min)a,b
Tc1[29]
Tc2
Tc3
Tc4
Tc5
17.80 (17.80)
16.83 (16.85)a
18.85 (18.82)a
17.76 (17.56)a
16.40 (16.35)a
15.50 (15.40)a
0.53 ꢃ 0.01
1.12 ꢃ 0.06
0.87 ꢃ 0.03
1.10 ꢃ 0.03
0.00 ꢃ 0.01
ꢀ0.02 ꢃ 0.03
23 ꢃ 2
36 ꢃ 2
74 ꢃ 2
87 ꢃ 3
48 ꢃ 4
62 ꢃ 3c
Tc6
a
Using a gradient of aqueous 0.1% CF3COOH and methanol as the solvent.
The values in parentheses are for the Re complexes Re2eRe7.
Concentration of melanin 0,5 mg/10 mL, 1 h incubation.
b
c
1 h with a suspension of synthetic melanin in distilled water, as
described elsewhere [28]. The percentage of binding, spanning
between 36 and 87%, depends very much on the donor-atom set
((S,N,O) vs (Npz,N,O) vs (Npy,N,O)) of the BFC used to stabilize the
organometallic core, being less influenced by the nature of the
pendant tertiary amine. The complexes with the (S,N,O)-donor
ligands, Tc2 and the previously reported Tc1 [29], presented the
lowest affinity to melanin with binding percentages of 36 ꢃ 2% and
23
ꢃ
2%, respectively. Tc3 and Tc4 anchored by pyrazolyl-
containing (N,N,O)-donor chelators have shown the highest
binding affinity with values of 74 ꢃ 2% and 87 ꢃ 3%, respectively.
Melanin is a polymer rich in negatively charged groups and
melanin-binders interact with this pigment mainly through ionic
interactions. However, such ionic interactions can be strengthened
by other attractions such as van der Waals and/or charge-transfer
processes involving the aromatic structures of the melanin-binders
and the indole monomers of melanin [16]. Probably, the occurrence
of charge-transfer processes between the pyrazolyl rings of Tc3 and
Tc4 and the indole groups of melanin can account for the higher
melanin affinity observed for these complexes.
Fig. 1. Cell uptake of Tc1eTc6 in murine B16-F1 melanoma cells and in human ame-
lanotic A375 melanoma cells, expressed as percentage of applied radioactivity per
million cells.
It has been shown that benzamide derivatives, particularly
those containing N-pyrrolidinyl substituents as is the case of Tc4,
2.2. Cell uptake studies
can recognize s-receptors [15,34e36], which are a class of proteins
existing in at least two subtypes (s1 and s2) [37]. The function of
these receptors is poorly understood but it is known that they are
over-expressed in different neoplastic tissues, namely in melanoma
Cellular uptake studies were performed for Tc1eTc6 in the
melanotic murine B16-F1 cell line versus the amelanotic A375
human cell line, in order to have an insight into the role of melanin
in the cellular accumulation of the complexes. The B16-F1 and A375
melanoma cells were incubated with the radioactive complexes at
different time points, up to 240 min. As can be seen in Fig. 1, the cell
uptake at 37 ꢁC was time-dependent and showed plateau values
spanning between 0.86 and 21.02% for the B16-F1 cell line and
between 0.49% and 13.58% for the A375 cell line, when expressed as
a percentage of total activity per million cells.
cell lines [15,36,37]. Hence, we decided to evaluate if s-receptors
could be involved in the cell uptake of the complexes reported
herein. Such evaluation has been performed only for the complexes
which presented a moderate cell uptake, i.e. Tc1, Tc3 and Tc4 in the
B16-F1 cell line and Tc4 in the A375 cell line, and involved
competition studies using haloperidol. Haloperidol is a non selec-
tive inhibitor of both s1 and s2 receptor subtypes [38]. It is well
known that the A375 amelanotic melanoma cell line expresses high
In the B16-F1 cell line, Tc1, Tc3 and Tc4 showed moderate
cellular uptake values (>10% at 4 h of incubation), being Tc4 the
complex with the best ability to accumulate in this cell line
(uptake of 21.02 ꢃ 0.71% at 4 h). In the amelanotic A375 cell line,
Tc4 has shown a moderate cell uptake (>10% at 4 h of incubation),
but all the other compounds have shown a relatively poor uptake,
i.e. inferior to 5%. Tc6 showed the lowest cell uptake in both cell
lines with almost negligible uptake values (<1% at 4 h of incu-
bation), which most probably reflects its hydrophilic character. All
tested complexes have shown a higher uptake in the melanotic
cell line compared to the amelanotic cell line. This finding might
indicate that the cellular retention of the compounds involves
their interaction with intracellular melanin. However, it is possible
that other intracellular targets could play a role in the cell accu-
mulation of the compounds, since Tc1 presented the lowest
levels of
the best of our knowledge, it has not been reported previously the
presence or not of -receptors in the B16-F1 cell line. Therefore, we
have evaluated the levels of s1-receptor expression in the B16-F1
cell line by Western blot analysis using a commercially available
antibody, as described in the experimental section. As a control, the
Western blot analysis was also performed for A375 and other tumor
cell lines (PC-3 [40] and MCF-7 [39,41]) that are known to express
s-receptors, as described in the literature [39]. However, to
s
s-receptors. The results obtained are presented in Fig. 2 and
confirmed the presence of a 30 KDa band corresponding to the s1
receptor, in all cell lines, including the B16-F1.
The difference in size between the band in the A375 cell line
sample and the band in the B16-F1 samples is due to the occurrence
of splicing variants of the receptor (which present slighty different
molecular weights) [37,40,41].
in vitro affinity for melanin (23
a reasonable cell uptake.
ꢃ
2%) but still displayed
As can be seen in Fig. 3, the incubation with haloperidol led to
a decrease of the cellular uptake of Tc1, Tc3 and Tc4 in the B16-F1

C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
355
cellular uptake of the 99mTc complexes in both cell lines is not due
to any alteration of the cell viability but probably results from the
blockade of
s receptors by haloperidol. These findings indicate that
the cellular uptake of these complexes may involve the interaction
with sigma receptors, particularly in the case of Tc4.
2.3. Biodistribution and imaging studies
The in vivo stability and the biodistribution of complexes
Tc1eTc6 were studied in B16-F1 melanoma-bearing mice, in order
to have a further insight into their relevance to design radioactive
probes for the targeting of melanotic melanoma.
The in vivo stability of Tc1eTc6 has been assessed by HPLC
analysis of the urine and blood of mice injected with these
complexes. These studies have shown that the intact complexes
correspond to the major part of the activity detected in the plasma
and in the urine, indicating that Tc1eTc6 are quite resistant to
metabolic transformation (Fig. S1).
The biodistribution data obtained for Tc1eTc6 at 1 h and 4 h p.i,
expressed in percent injected dose per gram tissue (%ID/g), are
presented in Table 2. All the complexes presented a relatively fast
blood clearance and a moderate rate of excretion, with low reten-
tion of radioactivity in non-target tissues, with the exception of the
organs involved in the excretion of the compounds, i.e. kidney, liver
and intestine. The biodistribution data indicate that Tc1eTc6
undergo both urinary and hepatobiliary excretion, being however
the hepatobiliar excretory pathway dominant as indicated by the
highest accumulation of activity in the intestine that spans
between 19.44 and 28.5 %ID/g at 4 h p.i.
Fig. 2. Evaluation of sigma 1 receptor expression in different cancer cell lines by
western blot analysis.
cell line for concentrations of haloperidol >10ꢀ9 M, being such
decrease more prominent for Tc4. Tc4 has shown an even more
pronounced decrease of cellular uptake in the A375 cell line, which
started for concentrations of haloperidol as low as 10ꢀ14 M (Fig. 3).
Haloperidol is a neuroleptic with potential toxic effects that can
affect the cell viability, as reported earlier for the M4 Beu cell line
[7]. Therefore, the changes on the cellular uptake of the 99mTc
complexes could arise from alterations in cell viability. To discard
this possibility, we have checked the eventual cytotoxic effects of
haloperidol against the B16-F1 and A375 cell lines, in the range of
haloperidol concentrations (10ꢀ14e10ꢀ4 M) used in the competi-
tion studies. The cell viability was evaluated after incubation with
different concentrations of haloperidol, at 37 ꢁC for 4 h, using
a colorimetric method based on the tetrazolium salt MTT ([3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]). The
MTT assays showed that the cytotoxic effects of haloperidol occur
uniquely for concentrations higher than 10ꢀ4 or 10ꢀ5 M for the B16-
F1 and A375 cell lines, respectively. Hence, the decrease of the
Complexes Tc1eTc6 showed low to moderate tumor uptake
values in the range 0.17 ꢃ 0.06 to 2.17 ꢃ 0.42 %ID/g at 1 h p.i. Tc5 and
Tc6, stabilized by pyridyl-containing BFCs, have shown the poorest
ability to target in vivo melanoma tissues in agreement with their
almost negligible in vitro cell uptake. Tc1 and Tc4, which have
shown the highest in vitro cell uptake, exhibited also the highest
tumor uptake with values of 2.17 ꢃ 0.42 and 1.74 ꢃ 0.24 %ID/g at 1 h
p.i., respectively. Tc1 has shown higher tumor retention than Tc4,
being observed a tumor uptake of 1.69 ꢃ 0.35 and 1.22 ꢃ 0.29 %ID/g
at 4 h p.i. for Tc1 and Tc4, respectively. At 1 h p.i., the tumor uptake
of Tc1 is roughly two-fold higher than the tumor uptake of the best
performing 99mTc(I) tricarbonyl complexes, containing the 4-
amino-N-(2-diethylaminoethyl)benzamide pharmacophore, previ-
ously reported by our research group [28].
The highest tumor uptake found for Tc1, together with its
favorable target/muscle ratio, prompted the use of this complex for
imaging studies in a B16-F1 melanoma-bearing mice. As can be
seen in Fig. 4, Tc1 clearly detected a melanoma tumor with low
retention of radioactivity by the surrounding non-target tissues.
However, a considerable accumulation of radioactivity was also
visualized in the gastrointestinal tract as expected from the bio-
distribution results.
3. Conclusion
Within our interest on 99mTc(I) organometallic complexes as
potential SPECT probes for in vivo imaging of melanotic melanoma,
we have evaluated a series of complexes (Tc1eTc6) with (S,N,O)-
and (N,N,O)-donor BFCs, bearing thioether, pyridyl or pyrazolyl
coordinating functions and functionalized with N-diethyl and N-
pyrrolidine groups for melanin binding. All the new 99mTc
complexes were obtained in high yield, and their chemical identity
was assessed by HPLC comparison with the Re congeners
(Re1eRe6). The ability of Tc1eTc6 to target melanoma cells, in vitro
or in vivo, is quite dependent on the nature of the BFCs. Tc5 and Tc6,
stabilized with the pyridyl-containing (N,N,O)-chelators, presented
Fig. 3. Effect of haloperidol concentration in the cell uptake of complexes Tc1, Tc3 and
Tc4 in murine B16-F1 melanoma cells and in the cell uptake of complex Tc4 in human
amelanotic A375 melanoma cells.

356
C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
Table 2
Relevant biodistribution data (% ID gꢀ1) of Tc1eTc6 at 1 and 4 h post-injection (p.i.) in C57/B16 mice (n ¼ 3e5) with palpable hind limb B16 melanoma nodules.
Mean tissue concentrations (%ID/g organ)
99mTc complex
Time (h, pi)
Muscle
Blood
Eyes
Tumor
Kidneys
Liver
Intestine
Excretion (% ID)
Tc1
1
4
1
4
1
4
1
4
1
4
1
4
0.42 ꢃ 0.07
0.10 ꢃ 0.01
0.37 ꢃ 0.05
0.05 ꢃ 0.01
0.88 ꢃ 0.08
0.16 ꢃ 0.08
0.88 ꢃ 0.24
0.15 ꢃ 0.02
0.63 ꢃ 0.22
0.22 ꢃ 0.06
0.51 ꢃ 0.13
0.28 ꢃ 0.06
0.87 ꢃ 0.13
0.40 ꢃ 0.05
0.68 ꢃ 0.21
0.25 ꢃ 0.05
0.99 ꢃ 0.17
0.41 ꢃ 0.12
0.82 ꢃ 0.21
0.26 ꢃ 0.08
0.48 ꢃ 0.13
0.25 ꢃ 0.06
0.79 ꢃ 0.15
0.33 ꢃ 0.08
1.54 ꢃ 0.15
1.41 ꢃ 0.27
1.38 ꢃ 0.30
0.82 ꢃ 0.56
1.71 ꢃ 0.40
1.11 ꢃ 0.19
1.34 ꢃ 0.24
1.24 ꢃ 0.10
0.56 ꢃ 0.05
0.63 ꢃ 0.19
0.89 ꢃ 0.17
1.49 ꢃ 1.05
2.17 ꢃ 0.42
1.69 ꢃ 0.35
1.44 ꢃ 0.32
0.89 ꢃ 0.38
0.99 ꢃ 0.19
0.79 ꢃ 0.76
1.74 ꢃ 0.24
1.22 ꢃ 0.29
0.97 ꢃ 0.15
0.81 ꢃ 0.20
0.42 ꢃ 0.06
0.27 ꢃ 0.05
2.39 ꢃ 0.13
1.32 ꢃ 0.34
1.64 ꢃ 0.17
0.67 ꢃ 0.23
3.92 ꢃ 0.95
1.10 ꢃ 0.26
7.21 ꢃ 2.09
1.35 ꢃ 0.28
4.77 ꢃ 0.68
2.53 ꢃ 0.35
3.10 ꢃ 0.52
1.81 ꢃ 0.23
4.96 ꢃ 0.40
3.66 ꢃ 0.69
3.53 ꢃ 0.80
1.36 ꢃ 0.24
5.46 ꢃ 1.54
2.86 ꢃ 1.03
3.72 ꢃ 0.53
2.19 ꢃ 1.09
4.24 ꢃ 0.64
2.31 ꢃ 0.45
6.29 ꢃ 0.69
5.08 ꢃ 1.30
20.59 ꢃ 1.36
19.44 ꢃ 1.38
22.92 ꢃ 2.49
17.55 ꢃ 4.07
25.91 ꢃ 1.51
23.41 ꢃ 2.78
26.80 ꢃ 2.06
28.56 ꢃ 2.15
18.74 ꢃ 1.9
21.39 ꢃ 2.04
17.39 ꢃ 0.53
21.84 ꢃ 6.71
31.6 ꢃ 11.1
56.9 ꢃ 7.2
Tc2
Tc3
Tc4
Tc5
Tc6
33.70 ꢃ 2.48
51.10 ꢃ 4.62
16.5 ꢃ 12.6
37.3 ꢃ 11.5
28.25 ꢃ 2.32
41.18 ꢃ 5.78
51.5 ꢃ 3.0
58.2 ꢃ 7.7
47.0 ꢃ 2.9
50.3 ꢃ 14.0
the lowest in vitro cell uptake and a very poor in vivo tumor uptake,
most probably due to the low log Do/w values of these complexes.
Tc1 and Tc4, containing respectively a cysteine-based (S,N,O)-
chelator and a pyrazolyl-containing (N,N,O)-chelator, have shown
the more favorable biological profile for in vivo targeting of mela-
notic melanoma, particularly Tc1 which has shown the highest
tumor uptake and encouraging target/non-target ratios. The
reasons for the highest tumor uptake observed for Tc1 are not clear,
since Tc1 presented the lowest binding affinity towards melanin
among all the tested compounds. An alternative to justify the best
presented a moderate tumor uptake and a relatively high accu-
mulation in the gastrointestinal tract. Therefore, similarly to other
99mTc complexes reported in the literature, Tc1 is not an alternative
to radioiodinated benzamides. Nevertheless, our results showed
that 99mTc complexes stabilized with a new (S,N,O)-donor BFC
derived from cysteamine have an enhanced ability to accumulate in
tumor cells, most probably due to their small size, lipophilicity and
favorable topology. These findings indicate that this new BFC
deserves to be further explored to obtain other 99mTc complexes
containing different small molecules directed against intracellular
targets with relevance for the detection of neoplastic tissues.
biological performance of Tc1 could be the involvement of
s
receptors. However, such possibility seems more probable for Tc4,
as shown by competitive studies with haloperidol. Tc1 allowed the
visualization of a melanotic melanoma in a tumor-bearing mice but
4. Experimental procedures
Unless otherwise stated, the synthesis and work-up of the
ligands and complexes were performed under air. The compounds
2-[2-(ethylthio)ethylamino]acetic acid [29], 2-[2-(3,5-dimethyl-
1H-pyrazol-1-yl)ethylamino]acetic acid [30], 2-[(pyridin-2-yl)
methylamino]acetic acid [31] and 2-[(2-(diethylamino)ethyl)(2-
(ethylthio)ethyl)amino]acetic acid (L1H) [29] were prepared
according to published methods. The starting material fac-
[Re(H2O)3(CO)3]Br [42] was synthesized by the literature method.
Na[99mTcO4] was eluted from a commercial 99Mo/99mTc generator,
using 0.9% saline. 1H and 13C NMR spectra were recorded on
a Varian Unity 300 MHz spectrometer; 1H and 13C chemical shifts
are given in ppm and were referenced with the residual solvent
resonances relative to SiMe4. IR spectra were recorded as KBr
pellets on a Bruker, Tensor 27 spectrometer. Electrospray ionisation
mass spectrometry (ESI-MS) was performed at ITN on a QITMS
instrument in positive ion mode. Thin-layer chromatography (TLC)
was performed on Merck silica gel 60 F254 plates. Column chro-
matography was performed with silica gel 60 (Merck). HPLC anal-
ysis of the Re and 99mTc complexes was performed on
a PerkineElmer LC pump 200 coupled to an LC 290 tunable UVeVis
detector and to a Berthold LB-507A radiometric detector, using an
analytical MachereyeNagel C18 reversed-phase column (Nucleosil
100e10, 250 ꢄ 3 mm) with a flow rate of 1 mL minꢀ1. HPLC puri-
fications were performed using a preparative Waters
m Bondapak
C18 column (150 ꢄ 19 mm) at a flow rate of 5.0 mL minꢀ1; UV
detection, 254 nm; eluents, A e aqueous 0.1% CF3COOH solution, B
e MeOH. The HPLC analysis and purifications were done with
gradient elution, using the following methods:
I) 0e5 min, 75% A; 5e30 min, 75%-50% A; 30e30.1 min, 50%-0%
A; 30.1e38 min 0% A.
II) 0e3 min, 100% A; 3e3.1 min, 100%-75% A; 3.1e9 min, 75% A;
9e9.1 min 75%-66% A; 9.1e20 min, 66%-0% A; 20e25 min, 0%
A; 25e25.1 min, 0%-100% A; 25.1e30 min, 100% A.
Fig. 4. Scintigraphic image of Tc1 in C57/B16 mice with palpable hind limb B16
melanoma nodules.

C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
357
III) 0e3 min, 100% A; 3e3.1 min, 100%-75% A; 3.1e9 min, 75% A;
4.4. Synthesis of 2-[(2-(diethylamino)ethyl)(pyridin-2-ylmethyl)
amino]acetic acid, L5
9e9.1 min 75%-66% A; 9,1e20 min, 66%-0% A; 20e25 min, 0%
A; 25e25.1 min, 0%-100% A; 25.1e30 min, 100% A.
L5H (24%, 200 mg) was prepared and purified as above
described for L2H, starting from 500 mg (3.01 mmol) of 2-[(pyridin-
2-yl)methylamino]acetic acid and 518 mg (3.01 mmol) of 2-chloro-
N,N-diethylethanamine.
4.1. Synthesis of 2-[(2-(ethylthio)ethyl)(2-(pyrrolidin-1-yl)ethyl)
amino]acetic acid, L2H
Rf (SiO2, CHCl3/MeOH/NH4OH (70/28/2)) ¼ 0.56; IR ymax/cmꢀ1
:
1594 (C^O); 1H NMR (CD3OD): dH 1.33 (6H, t, J ¼ 7.2 Hz, 2-CH3),
2.98 (2H, t, J ¼ 5.4 Hz, CH2), 3.15 (4H, q, J ¼ 5.5 Hz, 2 CH2), 3.20 (2H,
s, CH2), 3.22 (2H, m, CH2), 3.97 (2H, s, CH2), 7.34 (1H, t, J ¼ 6.0 Hz,
Hpy), 7.54 (1H, d, J ¼ 7.8 Hz, Hpy), 7.84 (1H, t, J ¼ 7.8 Hz, Hpy), 8.53
(1H, d, J ¼ 4.8 Hz, Hpy); 13C NMR (CD3OD): dc 7.93 (CH3), 46.92 (CH2),
50.94 (CH2), 52.11 (CH2), 58.11 (CH2), 60.38 (CH2), 122.98 (Cpy),
123.59 (Cpy), 137.83 (Cpy), 148.75 (Cpy), 158.50 (Cpy), 178.45 (C]O);
ESI/MS (þ) (m/z): 266.1 [M þ H]þ, calcd for C14H23N3O2 ¼ 265.2;
Anal. Calcd. for C14H22N3O2.NH4: C 59.49, H 9.29, N 19.85, found: C
59.27, H 9.04, N 19.84.
To a solution of 2-[2-(ethylthio)ethylamino]acetic acid (579 mg,
3.50 mmol) in H2O (15 mL) was added a solution of 1-(2-
chloroethyl)pyrrolidine (595 mg, 3.50 mmol) in THF (15 mL). The
pH was adjusted to 10e12 by addition of 5 M NaOH, and the
reaction mixture was refluxed overnight. The solvent was removed
under vacuum and the residue purified by silica gel column chro-
matography with CHCl3/MeOH/NH4OH (78/20/2) as eluent.
Compound L2H (16%, 145 mg) was recovered from the collected
fractions as a yellow oil, after removal of the solvent under reduced
pressure.
Rf (SiO2, CHCl3/MeOH/NH4OH (80/18/2)) ¼ 0.80; IR ymax/cmꢀ1
:
1587 (C^O); 1H NMR (CD3OD): dH 1.23 (3H, t, J ¼ 7.2 Hz, CH3), 2.09
(4H, m, 2 CH2), 2.60 (2H, q, J ¼ 7.2 Hz, CH2), 2.63 (2H, t, J ¼ 6.0 Hz,
CH2), 2.85 (4H, m, 2 CH2), 3.18 (2H, s, CH2), 3.19 (2H, m, CH2), 3.33
(4H, m, 2 CH2), 13C NMR (CD3OD): dc 13.35 (CH3), 21.92 (CH2), 24.96
(CH2), 28.55 (CH2), 51.02 (CH2), 52.38 (CH2), 52.50 (CH2), 56.00
(CH2), 57.05 (CH2), 178.02 (C]O); ESI/MS (þ) (m/z): 261.2 [M þ H]þ,
calcd for C12H26N2O2S ¼ 260.2; Anal. Calcd. for C12H23N2O2S.NH4: C
51.95, H 9.81, N 15.15, S 11.56, found: C 51.94, H 9.16, N 14.86, S 11.85.
4.5. Synthesis of 2-[(pyridin-2-ylmethyl)(2-(pyrrolidin-1-yl)ethyl)
amino]acetic acid, L6H
L6H (15%, 142 mg) was prepared and purified as above described
for L2H, starting from 600 mg (3.60 mmol) of 2-[(pyridin-2-yl)
methylamino]acetic acid and 612 mg (3.60 mmol) of 1-(2-
chloroethyl)pyrrolidine.
Rf (SiO2, CHCl3/MeOH/NH4OH (70/28/2)) ¼ 0.43; IR ymax/cmꢀ1
:
1595 (CO); 1H NMR (CD3OD): dH 2.10 (4H, m, 2-CH2), 2.99 (2H, t,
J ¼ 5.4 Hz, CH2), 3.20 (2H, s, CH2), 3.26 (2H, t, J ¼ 5.4 Hz, CH2), 3.33
(4H, m, 2 CH2), 3.95 (2H, s, CH2), 7.33 (1H, t, J ¼ 6.1 Hz, Hpy), 7.56
(1H, d, J ¼ 7.8 Hz, Hpy), 7.83 (1H, t, J ¼ 7.8 Hz, Hpy), 8.50 (1H, d,
J ¼ 4.8 Hz, Hpy); 13C NMR (CD3OD): dc 22.83 (CH2), 51.65 (CH2), 53.15
(CH2), 53.32 (CH2), 58.12 (CH2), 61.08 (CH2), 122.93 (Cpy), 123.84
(Cpy), 137.76 (Cpy), 148.72 (Cpy), 158.80 (Cpy), 178.36 (C]O); ESI/MS
(þ) (m/z): 264.1 [M þ H]þ, calcd for C14H21N3O2 ¼ 263,1; Anal.
Calcd. for C14H20N3O2.NH4: C 59.98, H 8.63, N 19.98, found: C 59.91,
H 8.97, N 19.71.
4.2. Synthesis of 2-[(2-(diethylamino)ethyl)(2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethyl)amino]acetic acid, L3H
L3H (36%, 85 mg) was prepared and purified as above described
for L2H, starting from 150 mg (0.76 mmol) of 2-[2-(3,5-dimethyl-
1H-pyrazol-1-yl)ethylamino]acetic acid and 131 mg (0.76 mmol) of
2-chloro-N,N-diethylethanamine.
Rf (SiO2, CHCl3/MeOH/NH4OH (78/20/2)) ¼ 0.61; IR ymax/cmꢀ1
:
1579 (CO); 1H NMR (CD3OD): dH 1.19 (6H, t, J ¼ 7.2 Hz, 2-CH3), 2.17
(3H, s, CH3-pz), 2.22 (3H, s, CH3-pz), 2.72 (2H, m, CH2), 2.87 (6H, m,
3 CH2), 3.02 (2H, t, J ¼ 5.7 Hz, CH2), 3.19 (2H, s, CH2), 4.02 (2H, t,
J ¼ 5.7 Hz, CH2), 5.83 (1H, s, H(4-pz)); 13C NMR (CD3OD): dc 8.08 (2
CH3), 10.95 (CH3-pz), 13.29 (CH3-pz), 46.94 (CH2), 48.73 (CH2),
49.87 (CH2), 53.41 (CH2), 54.27 (CH2), 58.40 (CH2), 106.09 (C(H4-
pz)), 142.37 (C(H3/5-pz)), 149.08 (C(H3/5-pz)), 179.65 (C]O); ESI/MS
(þ) (m/z): 297.0 [M þ H]þ calcd for C15H28N4O2 ¼ 296.2; Anal. Calcd.
for C15H27N4O2.NH4: C 57.48, H 9.97, N 22.34, found: C 57.59, H 9.47,
N 22.42.
4.6. General procedure for the synthesis of the Re complexes
(Re2eRe6)
[Re(H2O)3(CO)3]Br was reacted with equimolar amounts of
L2HeL6H in refluxing methanol for 18 h. After this time, the solvent
was removed under vacuum and the desired complexes were
purified by column chromatography or by RP-HPLC. In the case of
RP-HPLC, the purification was achieved using a preparative Waters
4.3. Synthesis of 2-[(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)(2-
m
Bondapak C18 (150 ꢄ 19 mm) with a flow rate of 5.0 mL minꢀ1
(pyrrolidin-1-yl)ethyl)amino]acetic acid, L4H
and using gradient elution with the methods indicated above for
each compound.
L4H (17%, 120 mg) was prepared and purified as above described
for L2H, starting from 480 mg (2.40 mmol) of 2-[2-(3,5-dimethyl-
1H-pyrazol-1-yl)ethylamino]acetic acid and 414 mg (2.40 mmol) of
1-(2-chloroethyl)pyrrolidine.
4.6.1. fac-[Re(CO)3(k3-L2)](Re2). Re2 (59%, 76 mg) was purified by
RP-HPLC using method I
IR ymax/cmꢀ1: 1677(CO), 1906, 2031 (C^O); 1H NMR (CD3OD):
T ¼ 20 ꢁC dH 1.34e1.45 (3H, m, CH3); 2.11 (4H, m, 2 CH2); 2.58-4.04
(16H, m); 13C NMR (CD3OD): T ¼ 20 ꢁC dc 12.10, 12.24 (CH3), 22.84,
28.71, 31.87, 32.27, 33.71, 49.63, 54.37, 54.64, 60.21, 60.82, 61.86,
63.49 (CH2), 114.31 (CTFA), 160.39 (CTFA), 180.72, 180.96 (C]O),
191.45, 191.80, 193.61, 195.22 (C^O); T ¼ 60 ꢁC dc 11.28 (CH3), 21.99
(CH2), 28.98 (br, CH2), 31.68, 31.99, 33.19 (br, CH2), 48.98 (CH2),
53.84 (CH2), 59.49 (CH2), 61.35, 62.45 (br, CH2), 63.40 (br, CH2),
114.27 (CTFA), 160.34 (CTFA), 180.67 (C]O), 190.76, 193.57 (C^O);
ESI/MS (þ) (m/z): 531.2 [M þ H]þ, calcd for ReC15H23N2O5S ¼ 530.1;
Anal. Calcd. for ReC15H23N2O5S. 2 CF3COOH: C 30.12, H 3.33, N 3.70,
S 4.23, found: C 30.32, H 4.34, N 4.05, S 4.43.
Rf (SiO2, CHCl3/MeOH/NH4OH (70/28/2)) ¼ 0.58; IR ymax/cmꢀ1
:
1584 (CO); 1H NMR (CD3OD): dH 2.00 (4H, m, 2 CH2), 2.17 (3H, s,
CH3-pz), 2.24 (3H, s, CH3-pz), 2.82 (2H, t, J ¼ 5.6 Hz, CH2), 3.07 (4H,
m, 2 CH2), 3.12 (2H, m, CH2), 3.28 (2H, t, J ¼ 7.2 Hz, CH2), 3.35 (2H, s,
CH2), 4.01 (2H, t, J ¼ 5.7 Hz, CH2), 5.84 (1H, s, H(4-pz)); 13C NMR
(CD3OD): dc 9.28 (CH3-pz), 12.13 (CH3-pz), 22.80 (CH2), 46.34 (CH2),
52.70 (CH2), 53.48 (CH2), 53.57 (CH2), 56.00 (CH2), 57.39 (CH2),
105.00 (C(H4-pz)), 141.20 (C(H3/5-pz)), 147.85 (C(H3/5-pz)), 178.79
(C]O); ESI/MS (þ) (m/z): 295.2 [M
þ
H]þ, calcd for
C15H26N4O2 ¼ 294.2; Anal. Calcd. for C15H27N4O2.NH4: C 57.85, H
9.39, N 22.49, found: C 57.79, H 9.07, N 22.42.

358
C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
4.6.2. fac-[Re(CO)3(k3-L3)](Re3). Re3 (61%, 42 mg) was purified by
RP-HPLC using method II
added to 400 mL of a solution of the organometallic precursor fac-
[
99mTc(H2O)3(CO)3]þ (1e2 mCi) in saline at pH 5.5. The reaction
IR ymax/cmꢀ1: 1675(CO); 1934, 2024 (C^O); 1H NMR (CD3OD): dH
1.37 (6H, t, J ¼ 7.2 Hz, 2-CH3), 2.33 (3H, s, CH3-pz), 2.48 (3H, s, CH3-
pz), 2.64 (2H, m, CH2), 3.45 (1H, m, CH2), 3.48 (4H, m, 2 CH2), 3.58
(3H, m, 2CH2), 3.84 (2H, m, 2CH2), 4.36 (2H, m, CH2), 6.16 (1H, s, H4-
pz); 13C NMR (CD3OD): dc 9.18 (CH3), 11.35 (CH3-pz), 15.95 (CH3-pz),
46.52 (CH2), 46.97 (CH2), 51.17 (CH2), 57.49 (CH2), 60.27 (CH2), 63.47
(CH2), 108.99 (C(H4-pz)), 115.47 (CTFA), 145.36 (C(H3/5-pz)), 155.50
(C(H3/5-pz)), 160.14 (CTFA), 180.77 (C]O), 195.58 (C^O), 196.61
(C^O), 197.35 (C^O); ESI/MS (þ) (m/z): 566.9 [M þ H]þ calcd for
ReC18H27N4O5 ¼ 566.2; Anal. Calcd. for ReC18H27N4O5.CF3COOH: C
35.24, H 4.15, N 8.24, found: C 35.16, H 4.45 N 8.37.
mixture was then heated to 100 ꢁC for 30 min, cooled to room
temperature and the pH adjusted to pH ¼ 7.4. The final solutions
were analyzed by RP-HPLC using a MachereyeNagel C18 reversed-
phase column (Nucleosil 10
m
m, 250 ꢄ 4 mm) and a gradient of
aqueous 0.1% CF3COOH (A) and methanol (B) with a flow rate of
1.0 mL minꢀ1 and using method III.
4.8. Determination of distribution coefficients
The log Do/w values of complexes Tc1eTc6 were determined by
the ‘‘shake flask’’ method under physiological conditions (n-
octanole0.1 M PBS, pH 7.4) [33].
4.6.3. fac-[Re(CO)3(k3-L4)](Re4). Re4 (79%, 83 mg) was purified by
RP-HPLC using method I
4.9. In vitro binding to melanin
IR ymax/cmꢀ1: 1677 (CO), 1901, 2026 (C^O), 1H NMR (CD3OD):
dH 2.03 (4H, m, 2CH2), 2.33 (3H, s, CH3-pz), 2.48 (3H, s, CH3-pz), 2.59
(2H, m, CH2), 3.35 (1H, m, CH2), 3.47 (4H, m, 2 CH2), 3.53 (1H, m,
CH2), 3.74 (2H, m, CH2), 3.82 (1H, m, CH2), 3.90 (1H, m, CH2), 4.35
(2H, m, CH2), 6.16 (1H, s, H4-pz); 13C NMR (CD3OD): dc 9.34 (CH3-pz),
13.97 (CH3-pz), 22.01 (CH2), 44.53 (CH2), 48.15 (CH2), 53.76 (CH2),
55.20 (CH2), 58.83 (CH2), 61.48 (CH2), 106.93 (C(H4-pz)), 116.27
(CTFA), 143.37 (C(H3/5-pz)), 153.44 (C(H3/5-pz)), 160.14 (CTFA), 178.86
(C]O), 192.85 (C^O), 193.87 (C^O), 194.48 (C^O); ESI/MS (þ) (m/
z): 565.2 [M þ H]þ, calcd for ReC18H25N4O5 ¼ 564,1; Anal. Calcd. for
ReC18H25N4O5.2CF3COOH: C 33.38, H 3.44, N 7.08, found: C 33.26, H
3.45, N 7.37.
The binding affinity to melanin of Tc1eTc6 was assessed using
synthetic tyrosineemelanin (SigmaeAldrich, St Louis, Missouri,
USA). The general procedure used was as follows: A 100 mL aliquot
of the radioactive preparations of Tc1eTc6 was added to a melanin
suspension (0.5 mg/10 ml) in distilled water. The reaction mixture
was incubated at room temperature for 1 h with stirring. After
incubation, the samples were centrifuged at 30,000 g for 10 min,
and aliquots of the supernatant were counted in the gamma
counter. Control samples were also used containing the radioactive
preparation without melanin. The difference between the activity
of aliquots from the supernatants of the test samples (with
melanin) and the control samples (without melanin) allowed the
calculation of the percentage of unbound complexes.
4.6.4. fac-[Re(CO)3(k3-L5)](Re5). Re5 (55%, 50 mg) was purified by
silica-gel column chromatography using CHCl3/MeOH/NH4OH (70/
28/2) as eluent
Rf (SiO2 and CHCl3/MeOH/NH4OH (70/28/2)) ¼ 0.72.IR ymax/cmꢀ1
:
4.10. Cell culture
1652 (CO), 1876, 2019 (C^O); 1H NMR (CD3OD): dH 1.10 (6H, t,
J ¼ 7.2 Hz, 2-CH3), 2.68 (4H, m, 2 CH2), 2.94 (2H, m, CH2), 3.49 (1H, d,
J ¼ 17.1 Hz, CH2), 3.70 (2H, m, CH2), 4.03 (1H, d, J ¼ 17.1 Hz, CH2), 4.60
(1H, d, J ¼ 15.6 Hz, CH2), 4.76 (1H, d, J ¼ 15.6 Hz, CH2), 7.53 (1H, t,
J ¼ 6.0 Hz, Hpy), 7.69 (1H, d, J ¼ 7.8 Hz, Hpy), 8.08 (1H, t, J ¼ 7.8 Hz, Hpy),
8.80 (1H, d, J ¼ 4.8 Hz, Hpy); 13C NMR (CD3OD): dc 11.69 (CH3), 48.12
(CH2), 50.17 (CH2), 62.09 (CH2), 68.05 (CH2), 69.64 (CH2),125.00 (Cpy),
127.00 (Cpy), 141.69 (Cpy), 153.55 (Cpy), 160.72 (Cpy), 183.17 (C]O),
195.63 (C^O), 196.39 (C^O), 196.61 (C^O); ESI/MS (þ) (m/z): 536.0
[M þ H]þ, calcd for ReC17H22N3O5 ¼ 535.0; Anal. Calcd. for
ReC17H22N3O5: C 38.20, H 4.15, N 7.86, found: C 38.52, H 4.34, N 7.62.
B16-F1 murine melanotic melanoma (ECACC, UK) and A375
human amelanotic melanoma cells (ATCC, Spain) were grown in
DMEM containing GlutaMax I supplemented with 10% heat-
inactivated fetal bovine serum and 1% penicillin/streptomycin
antibitiotic solution (all from Invitrogen). Cells were cultured in
a humidified atmosphere of 95% air and 5% CO2 at 37 ꢁC (Heraeus,
Germany), with the medium changed every other day. The cells
were adherent in monolayers and, when confluent, were harvested
from the cell culture flasks with trypsineEDTA and seeded farther
apart.
4.6.5. fac-[Re(CO)3(k3-L6)](Re6). Re6 (39%, 55 mg) was purified by
RP-HPLC using method I
4.11. Cellular uptake studies and cytotoxicity assays
IR ymax/cmꢀ1: 1679(CO), 1906, 2029 (C^O); 1H NMR (CD3OD):
dH 2.13 (4H, m, 2-CH2), 3.57 (1H, d, J ¼ 17.1 Hz, CH2), 3.90 (6H, m,
3CH2), 4.03 (3H, m, 2 CH2), 4.65 (1H, d, J ¼ 15.3 Hz, CH2), 4.80 (1H, d,
J ¼ 15.3 Hz, CH2), 7.58 (1H, t, J ¼ 6.3 Hz, Hpy), 7.72 (1H, d, J ¼ 7.8 Hz,
Hpy), 8.15 (1H, t, J ¼ 7.8 Hz, Hpy), 8.83 (1H, d, J ¼ 5.1 Hz, Hpy); 13C
NMR (CD3OD): dc 22.01 (CH2), 48.83 (CH2), 53.82 (CH2), 59.53 (CH2),
62.55 (CH2), 67.12 (CH2), 115.34 (CTFA), 123.19 (Cpy), 125.30 (Cpy),
139.94 (Cpy), 151.60 (Cpy), 157.92 (Cpy), 160.02 (CTFA), 180.38 (C]O),
194.85 (C^O), 195.49 (C^O), 195.71 (C^O); ESI/MS (þ) (m/z):
534.2 [M þ H]þ, calcd for ReC17H20N3O5 ¼ 533.1; Anal. Calcd. for
ReC17H20N3O5. 3CF3COOH: C 31.55, H 2.76, N 4.80, found: C 31.53, H
4.89, N 2.91.
Cellular uptake assays of the 99mTc complexes (Tc1eTc6) were
performed in B16-F1 and A375 cells seeded at a density of 0.2
million/0.5 mL culture medium per well in 24-well tissue culture
plates and allowed to attach overnight. After that period, the
medium was removed and replaced by fresh medium containing
approximately 2 ꢄ 105cpm/0.5 mL of each 99mTc complex. The cells
were incubated again under humidified 5% CO2 atmosphere, at
37 ꢁC for a period of 15 min to 4 h. After 0.25, 0.5, 1, 2, 3 and 4 h
incubation period the cells were washed twice with cold PBS, lysed
with 0.1 M NaOH and the cellular extracts were counted for
radioactivity. Each experiment was performed in quadruplicate.
Cellular uptake data were expressed as an average value plus the
standard deviation.
4.7. Synthesis of the 99mTc(I) complexes (Tc1eTc6)
The cell uptake of complexes Tc1, Tc3 and Tc4 in B16-F1 murine
melanoma and A375 human amelanotic melanoma cells was also
evaluated in competition experiments with haloperidol. In these
competition experiments intact cells were incubated with the test
complex in the presence of different concentrations of haloperidol
The 99mTc complexes were obtained as previously described for
Tc1 [29], using the following general method: In a nitrogen-purged
glass vial, 40 m
L of a 4.4 x 10ꢀ5 M aqueous solution of L1HeL6H were

C. Moura et al. / European Journal of Medicinal Chemistry 50 (2012) 350e360
359
(10ꢀ14 to 10ꢀ4 M) for 4 h. After this time, the cell uptake was
measured as described above.
interest were dissected, rinsed to remove excess blood, weighed,
and their radioactivity was measured using a -counter (LB2111,
g
The cytotoxicity of haloperidol against the two different cell lines
was evaluated using a colorimetric method based on the tetrazolium
salt MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide), which is reduced by viable cells to yield purple for-
mazan crystals. Cells were seeded in 96-well plates at a density of
50 x 105 cells per well, allowed to attach overnight and then incu-
bated for 4 h in the presence of various concentrations of haloper-
idol, dissolved in ethanol (the maximum percentage of ethanol did
not exceed 2%). At the end of the incubation period the medium was
removed and the cells were incubated with MTT (0.5 mg/mL in
Berthold, Germany). The uptake in the tumor and healthy tissues
was calculated and expressed as a percentage of the injected
radioactivity dose per gram of tissue. For blood, bone, muscle, and
skin, total activity was estimated assuming that these organs
constitute 6, 10, 40, and 15% of the total body weight, respectively.
Urine was also collected and pooled together at the time the
animals were killed.
For imaging, the animals were injected with complex Tc1
(29 MBq) and sacrificed at 1 h p.i.. A set of static images (256 ꢄ 256
matrix, Zoom 2, 2 min) were acquired, by placing the animals over
culture medium; 200
formazan crystals formed inside the cells were then dissolved in
200 l of DMSO by thorough shaking, and the absorbance was read
m
l) for 3 h at 37 ꢁC and 5% CO2. The purple
a g camera (GE 400AC; Maxicamera, Milwaukie, USA) coupled with
a high-resolution parallel collimator and controlled with GENIE
Acquisition computer.
m
at 570 nm, using a plate spectrophotometer (Power Wave Xs; Bio-
Tek). Each test was performed with at least six replicates and
repeated at least 2 times. The cell viability was calculated dividing
the absorbance of each well by that of the control wells (cells treated
with medium and without addition of haloperidol).
4.14. In vivo stability studies
The in vivo stability of Tc1-Tc6 was evaluated by urine and
murine serum HPLC analysis, using the elution conditions above
described for the analysis of these 99mTc complexes. The urine was
collected at sacrifice time and filtered through a Millex GV filter
4.12. Evaluation of Sigma-1 receptor expression
(0.22
immediately centrifuged for 15 min at 3000 rpm at 4 ꢁC, and the
serum was separated. Aliquots of 100 L of serum were treated with
200
mm) before RP-HPLC analysis. Blood collected from mice was
Western blot experiments were performed to evaluate the levels
of expression of Sigma-1 receptor in the following cell lines: A375,
PC-3, MCF-7 and B16-F1. Cells were lysed in Cell Lytic-MT Extrac-
tion reagent (Sigma) supplemented with Complete protease
inhibitor cocktail tablets (Roche). After 15 min on ice, lysates were
centrifuged at 14,000 g for 10 min at 4 ꢁC to pellet the cellular
debris and the supernatants removed for further use. The total
protein content was determined by using the DC Protein Assay Kit
m
m
L of ethanol to precipitate the proteins. Samples were
centrifuged at 4000 rpm for 15 min, at 4 ꢁC. Supernatant was
collected and passed through a Millex GV filter (0.22 lm) prior to
RP-HPLC analysis.
Acknowledgements
(Biorad) and aliquots of protein (30
analyzed using standard western blot procedures. Briefly, protein
extracts were subjected to electrophoresis on 10% SDS-
mg) from each sample were
Carolina Moura thanks the FCT for a Doctoral research grant
(SFRH/BD/38469/2007). J. Marçalo is acknowledged for the ESI-MS
analyses which were run on a QITMS instrument acquired with the
support of the Programa Nacional de Reequipamento Científico
(Contract REDE/1503/REM/2005 - ITN) of FCT and is part of RNEM -
Rede Nacional de Espectrometria de Massa.
a
polyacrylamide gel and transferred electrophoretically onto nitro-
cellulose membranes. The blots were blocked with (PBS þ 0.1%
Tween20) containing 5% nonfat dry milk for 1 h. Then, the blotting
membranes were incubated with primary antibodies against
Sigma-1 (1:200, goat polyclonal, Abcam) and actin (1:8000, mouse
monoclonal, Sigma) overnight. Membranes were washed with PBS-
T and incubated for 1 h with secondary antibody (donkey anti-goat
IgG-HRP, Santa Cruz Biotechnologyand goat anti-mouse IgG-HRP,
Biorad) diluted 1:3000. Finally, membranes were developed using
the SuperSignal WetsPico Substrate kit (Pierce, Rockford, IL)
according to the manufacturer’s instructions.
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.02.014.
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