Mono- and dicationic Re(I)/99mTc(I) tricarbonyl complexes for the targeting of energized mitochondria

Article abstract of DOI:10.1016/j.jinorgbio.2013.02.008

The enhanced negative mitochondrial membrane potential of tumor cells can increase the cell accumulation of triphenylphosphonium (TPP) derivatives, which prompted us to investigate TPP-containing Re(I)/^99mTc organometallic compounds as probes for in vivo targeting of energized mitochondria. Novel compounds (Re1-Re4/Tc1-Tc4) were obtained with bifunctional chelators of the pyrazole-diamine (N,N,N-donors) and pyrazole-aminocarboxylic (N,N,O-donors) type, functionalized with TPP pharmacophores that have been introduced at the central amine of the chelators using different spacers. In this way, dicationic (Re1-Re2, Tc1-Tc2) and monocationic (Re3-Re4, Tc3-Tc4) complexes with variable lipophilicity were synthesized. The ^99mTc complexes (Tc1-Tc4) are highly stable under physiological conditions and their chemical identification was done by HPLC comparison with the Re congeners (Re1-Re4), which were fully characterized by common analytical techniques (electrospray ionization mass spectrometry (ESI-MS), IR, multinuclear NMR). The in vitro biological evaluation of Tc1-Tc4 was performed in a panel of human tumor cell lines (PC-3, MCF-7 and H69), including cell lines overexpressing P-glycoprotein (MCF-7/MDR1 and H69/Lx4), and in isolated mitochondria. All the tested complexes showed a low to moderate cellular and mitochondrial uptake and did not undergo significant P-glycoprotein (Pgp)-mediated efflux processes. In particular, the dication Tc2 and the monocation Tc4 presented the highest cellular and mitochondrial uptake. Their cellular uptake was shown to depend on the mitochondrial (Δψ_m) and plasma membrane (Δψ_p) potentials. Altogether, the biological properties of these compounds suggest that they might be relevant for the design of radioactive metalloprobes for in vivo targeting of mitochondria.

Full text of DOI:10.1016/j.jinorgbio.2013.02.008

Journal of Inorganic Biochemistry 123 (2013) 34–45
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Mono- and dicationic Re(I)/99mTc(I) tricarbonyl complexes for the targeting of
energized mitochondria
⁎
Carolina Moura, Filipa Mendes, Lurdes Gano, Isabel Santos, António Paulo
Unidade de Ciências Químicas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Instituto Superior Técnico, Universidade Técnica de Lisboa, Estrada Nacional 10, 2686-953,
Sacavém, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 January 2013
Received in revised form 11 February 2013
Accepted 12 February 2013
Available online 26 February 2013
The enhanced negative mitochondrial membrane potential of tumor cells can increase the cell accumulation
of triphenylphosphonium (TPP) derivatives, which prompted us to investigate TPP-containing Re(I)/99mTc
organometallic compounds as probes for in vivo targeting of energized mitochondria. Novel compounds
(Re1–Re4/Tc1–Tc4) were obtained with bifunctional chelators of the pyrazole-diamine (N,N,N-donors)
and pyrazole-aminocarboxylic (N,N,O-donors) type, functionalized with TPP pharmacophores that have been
introduced at the central amine of the chelators using different spacers. In this way, dicationic (Re1–Re2,
Tc1–Tc2) and monocationic (Re3–Re4, Tc3–Tc4) complexes with variable lipophilicity were synthesized. The
99mTc complexes (Tc1–Tc4) are highly stable under physiological conditions and their chemical identification
was done by HPLC comparison with the Re congeners (Re1–Re4), which were fully characterized by common
analytical techniques (electrospray ionization mass spectrometry (ESI-MS), IR, multinuclear NMR). The in
vitro biological evaluation of Tc1–Tc4 was performed in a panel of human tumor cell lines (PC-3, MCF-7 and
H69), including cell lines overexpressing P-glycoprotein (MCF-7/MDR1 and H69/Lx4), and in isolated
mitochondria. All the tested complexes showed a low to moderate cellular and mitochondrial uptake and did
not undergo significant P-glycoprotein (Pgp)-mediated efflux processes. In particular, the dication Tc2 and the
monocation Tc4 presented the highest cellular and mitochondrial uptake. Their cellular uptake was shown
to depend on the mitochondrial (Δψm) and plasma membrane (Δψp) potentials. Altogether, the biological
properties of these compounds suggest that they might be relevant for the design of radioactive metalloprobes
for in vivo targeting of mitochondria.
Keywords:
Rhenium
Technetium-99m
Tricarbonyl
Mitochondria
Triphenyphosphonium derivatives
© 2013 Elsevier Inc. All rights reserved.
1. Introduction
features enable the use of SPECT or PET to visualize interactions
between physiological targets and adequate ligands, allowing their
Mitochondria are dynamic organelles that play a central role in
cellular metabolism. In recent years, there has been an increasing
awareness that defects in mitochondrial function contribute to the
development and progression of cancer. Several distinct differences
between the mitochondria of normal cells and cancer cells are
known to occur at the genetic, molecular and biochemical levels,
offering the possibility of exploring mitochondria as targets for novel
and site-specific anti-cancer agents [1–4]. These differences also render
mitochondria suitable markers for the early detection of cancer, based
on molecular imaging techniques.
Among the existing molecular imaging modalities (e.g. nuclear
imaging, magnetic resonance imaging, computed tomography, ultra-
sound, bioluminescence and fluorescence imaging), the nuclear tech-
niques single photon emission computed tomography (SPECT) and
positron emission tomography (PET) have the advantage of high
intrinsic sensitivity and unlimited depth penetration. These favorable
application in the clinical setting. To fully profit from the intrinsic
advantages of SPECT and PET it is necessary to design radioprobes
that are able to accumulate in a given target tissue upon specific
interaction with a given biomarker of disease, such as an up-regulated
membranar receptor or an altered organelle like the energized mito-
chondria of tumor cells.
The rationale behind the use of radioprobes to target mitochondria
of tumor cells relies on the increased negative membrane potential
(Δψm) across the inner membrane of these organelles in cancer cells
compared with normal cells [5–8]. Several research groups have used
triphenylphosphonium (TPP) derivatives in the design of radioprobes
for in vivo targeting of mitochondria [8–17]. The use of TPP derivatives
to design this type of probes has been driven by the ability of 3H-
triphenylphosphonium and 3H-methyltriphenylphosphonium to accu-
mulate in mitochondria and to measure mitochondrial potentials
[10–12,14]. These lipophilic and delocalized cationic molecules pene-
trate the hydrophobic barriers of the plasma and mitochondrial mem-
branes and accumulate inside the energized mitochondria because of
their positive charge [14,18]. Since cancer cells have an increase of
⁎
Corresponding author. Tel.: +351 219946196.
E-mail address: apaulo@itn.pt (A. Paulo).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.02.008

C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
35
approximately 60 mV in Δψm (i.e., more negative), this difference alone
is sufficient to account for a ten-fold accumulation of these compounds
in carcinoma cells vs normal cells, in accordance with the Nernst equa-
tion [14,18]. The existence of a negative plasma membrane potential
(Δψp) (−30 to −60 mV) further concentrates lipophilic cations in the
cytosol, contributing also for their increased accumulation in tumoral
mitochondria.
of cellular uptake in several human tumor cell lines, including Pgp-
expressing lines, uptake studies in isolated mitochondria, and the
assessment of the influence of Δψm and Δψp in cellular uptake.
2. Results and discussion
2.1. Chemistry
The major part of the work on radiolabeled TPP derivatives has
involved PET radioisotopes, such as 18F and 64Cu. Encouraging results
were found in terms of selectivity for tumor mitochondria, but unfa-
vorable biodistribution profiles were reported for most of the com-
pounds due to undesirable accumulation in non-target soft tissues
[9,13,15–17]. The use of SPECT rather than PET tracers has the poten-
tial advantage of wider availability at lower costs. This is particularly
true for 99mTc-based radioprobes, since the 99mTc radionuclide still is
the “work horse” for SPECT imaging in clinical nuclear medicine [19].
Despite these advantages, the efforts made to obtain 99mTc-labelled
TPP derivatives for mitochondria targeting are scarce. To the best of
2.1.1. Synthesis and characterization of the ligands
As mentioned above, we have explored bifunctional chelators of
the pyrazole-diamine (N,N,N-donors) and pyrazole-aminocarboxylic
(N,N,O-donors) type to obtain Re(I)/99mTc(I) tricarbonyl complexes
functionalized with TPP derivatives [23–25]. The use of these chelators
has been motivated by the possibility of obtaining structurally related
Re(I)/99mTc(I) tricarbonyl with different lipophilicity and different
overall charge, which can be determinant factors of their mitochondria
targeting capability. Moreover, these two classes of chelators allowed
the functionalization with TPP derivatives using convergent synthetic
approaches.
our knowledge, only
a mercaptoacetyltriglycine (MAG3) Tc(V)
oxocomplex bearing a TPP derivative has been evaluated as a SPECT
probe for the targeting of tumoral mitochondria [20]. However, the
overall charge of this Tc(V) complex is neutral, which is a potential
drawback for the desired purpose. It is worth mentioning that
As shown in Schemes 1 and 2, the functionalization of both classes
of chelators with the TPP derivatives was done using a common
synthetic approach, starting from compounds 1 and 4, respectively.
This approach involved the N-alkylation of the central amine of 1
and 4 with (4-bromobutyl)triphenylphosphonium bromide or (4-
bromomethyl)benzyl)triphenylphosphonium bromide [13], carried
out in DMF (dimethyl formamide) and in the presence of
triethylamine. These N-alkylation reactions afforded compounds 2
and 3 (Scheme 1) and compounds 5 and 6 (Scheme 2), respectively,
functionalized with the TPP derivatives at the central amine group.
The removal of the tert-butyloxycarbonyl (BOC) protecting group
from 2 and 3 with TFA gave the final ligands L1 and L2, respectively,
which were obtained as trifluoroacetate salts in moderate-to-high
yield. In a first attempt, the hydrolysis of the ester function of 5 and
6 was done under basic conditions but the reaction proceeded with
the oxidation of the phosphonium group. As indicated in Scheme 2,
the use of concentrated HCl avoided the oxidation reaction and led
to an efficient hydrolysis of the ester function, yielding the final
chelators L3H and L4H, respectively. L3H and L4H were obtained as
zwitterions in moderate yield, after neutralization of the reaction
mixture and desalting with a C18 Sep-Pak cartridge that has been
eluted successively with distilled water and methanol. The character-
ization of the new phosphonium-containing ligands (L1–L4H) by
multinuclear NMR (1H, 13C and 31P), electrospray ionization mass
spectrometry (ESI-MS), IR and elemental analysis confirmed the
respective formulations.
[
99mTcI(MIBI)6]+ 99mTc-Sestamibi) and [99mTcV(tetrofosmin)2O2]+
( ,
in clinical use as myocardial perfusion imaging agents, have also
relevance for tumoral detection due to their ability to accumulate in
the mitochondria of tumor cells. Unfortunately, these two complexes
are also substrates of multidrug resistance P-glycoprotein (PgP),
which may induce a rapid efflux of these cationic and lipophilic radio-
tracers from tumor cells [19,21,22].
Due to the versatility and unique features of the so-called
tricarbonyl approach, the use of the fac-[99mTc(CO)3]+ core can easily
afford complexes of different charge with tunable lipophilicity. We
decided to study Tc(I) organometallic complexes bearing TPP deriva-
tives aiming at the design of SPECT probes for in vivo targeting of
mitochondria. To achieve this goal, we have explored pyrazole-diamine
(N,N,N-donors) and pyrazole-aminocarboxylic (N,N,O-donors) chelators
[23–25]. These chelators were functionalized with TPP derivatives and
used to synthesize structurally related mono- and dicationic complexes,
as depicted in Fig. 1. By using compounds possessing an intrinsic +2
charge vs a +1 charge we expected to obtain a further enhancement
(i.e. a 100-fold vs 10-fold) of the mitochondrial accumulation in carcino-
ma cells vs normal cells, according to the Nernst equation [14].
Herein, we report on the synthesis, characterization and in vitro
evaluation of new 99mTc(I)/Re(I) tricarbonyl complexes (Tc1–Tc4/
Re1–Re4), as probes for in vivo targeting of mitochondria. To gain an
insight into their relevance for this type of application, the in vitro bio-
logical evaluation of the 99mTc complexes comprised the measurement
2.2. Synthesis and characterization of the Re and 99mTc complexes
Non-radioactive Re complexes are commonly used as surrogates of
the 99mTc congeners, profiting from the physico-chemical similarities
of these group 7 elements. In this way, one can avoid the use of the
long-lived β− emitter 99Tc available in macroscopic amounts for the
synthesis and structural analysis of technetium compounds. For L1–
L4H, the respective Re complexes (Re1–Re4) were prepared with the
goal of using them as surrogates of the 99mTc congeners, to assign
their chemical identity by means of HPLC comparison. Re1–Re4 were
obtained in low to moderate yield (24–59%) by reacting fac-
[Re(H2O)3(CO)3)]Br with the corresponding ligands in refluxing meth-
anol or H2O (Scheme 3). Their purification was done by washing with
n-hexane and water (Re1) or by semi-preparative reverse-phase
(RP)-HPLC (Re2–Re4).
Complexes Re1–Re4 were characterized by elemental analysis,
ESI-MS and by common spectroscopic techniques (IR; 1H, 31P and
13C NMR). The positive ESI-MS spectra of Re1–Re4 showed prominent
peaks corresponding to the expected molecular-ions, with the correct
isotope distribution pattern and without significant fragmentation.
Fig. 1. General molecular structures of the Re and 99mTc complexes evaluated as probes
for the targeting of mitochondria.

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C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
Scheme 1. Synthesis of L1 and L2. Reagents and conditions: (i) Br(CH2)4P+Ph3, DMF, NEt3, r.t., 72 h; (ii) Br-p-xylene-P+Ph3, DMF, NEt3, r.t., 72 h; and (iii) TFA, CH2Cl2, r.t., 2.5 h.
The IR spectra of Re1–Re4 showed a typical fac-[Re(CO)3]+ pattern
with the presence of two strong ν(C`O) bands centered at 1896 and
2027 cm−1, displaying frequencies comparable to those found for
other Re(I) tricarbonyl complexes anchored by the same type of chela-
tors [24,26]. The 1H NMR spectra of Re1–Re4 presented a set of multi-
plets for the diastereotopic methylenic protons of the framework of
the pyrazolyl-diamine or pyrazolyl-aminocarboxylic ligands. The
patterns of the 1H NMR spectra were consistent with a facial coordina-
tion of the chelators, through the pyrazolyl ring, the central nitrogen
atom and the terminal amine (Re1 and Re2) or carboxylate (Re3 and
Re4) groups, as previously reported for related complexes [24–26].
The presence of three C`O resonances, spanning from 193.66 to
196.85 ppm, in the 3C NMR spectra of Re1–Re4 also corroborated the
tridentate coordination of the chelators. The presence of a unique
Scheme 2. Synthesis of L3H and L4H. Reagents and conditions: (i) Br(CH2)4P+Ph3, DMF, NEt3, r.t., 72 h; (ii) Br-p-xylene-P+Ph3, DMF, NEt3, r.t., 72 h; (iii) HCl 4 M, reflux, 4 h.

C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
37
Scheme 3. Synthesis of the Re (Re1–Re4) and 99mTc (Tc1–Tc4) complexes.
singlet in the corresponding 31P{1H} spectra, between 24.05 and
24.64 ppm, confirmed the presence of the TPP groups, and showed
that these groups were not oxidized during the synthesis of the
complexes.
by (N,N,O)-donor BFCs, exhibited a lipophilic character with log Do/w
values ranging from 0.55 to 0.79. For the complexes with the
same donor atom set, replacement of the butylene (Tc1 and Tc2) by a
p-xylene (Tc3 and Tc4) linker resulted in an increase of lipophilicity.
The 99mTc congeners (Tc1–Tc4) were obtained in aqueous solu-
tion, at pH 7.4 (Tc1 and Tc2) or pH 5.5 (Tc3 and Tc4), by reaction of
fac-[99mTc(H2O)3(CO)3]+ with the appropriate ligand at 100 °C for
30 min (Scheme 3). Unlike Tc2, all the other 99mTc complexes were
obtained in almost quantitative yield (≥95%) using ligand concentra-
tions as low as 10−4 M. Tc2 was synthesized with a moderate yield
(70%) due to the formation of an unidentified radiochemical impurity.
For this reason, Tc2 was purified by semi-preparative RP-HPLC, being
obtained with a radiochemical purity >99% after purification. Only
the purified Tc2 was used in the in vitro studies described below.
The chemical identity of Tc1–Tc4 was established by comparing
their HPLC radiochromatograms with the UV–visible (UV–vis) trace
of the fully characterized Re surrogates, as exemplified for Tc4/Re4
in Fig. 2. The lipophilicity of the 99mTc complexes was assessed by
measurement of the respective log Do/w values (n-octanol/0.1 M,
phosphate buffered saline (PBS), pH 7.4) using the multiple “shake-
flask” method. The log Do/w values and the HPLC retention times of
Tc1–Tc4 are shown in Table 1. The dicationic complexes Tc1 and Tc2,
anchored by (N,N,N)-donor pyrazolyl-diamine bifunctional chelators
(BFCs), are hydrophilic with log Do/w values of −0.93
0.07 and
Fig. 2. HPLC chromatograms of co-injected rhenium complex (Re4) (UV detection) and
99mTc complex (Tc4) (γ detection).
−0.07 0.05, respectively. The monocationic Tc3 and Tc4, anchored

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C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
Table 1
effect of Δψp and Δψm on the cellular uptake has been also evaluated
by depolarization of the cellular and/or mitochondrial membrane
potentials.
HPLC Retention times and log D7.4 of complexes Tc1–Tc4.
Complex
rt (min)a,b
log D7.4
Tc1
Tc2
Tc3
Tc4
21.18 (21.08)
20.67 (20.49)
21.78 (21.60)
21.89 (21.68)
−0.93
−0.07
0.59
0.07
0.05
0.05
0.01
3.1. Cellular uptake
0.74
The first screening of the ability of complexes Tc1–Tc4 to accumu-
late in human tumor cells has been performed using PC-3 prostate
cancer cells and MCF-7 breast cancer cells. The cellular uptake of
Tc1–Tc4 at 37 °C was time-dependent in both cell lines (Fig. 3A and
B) with values in the ranges 3.47–8.30% and 3.12–4.84% for the PC-3
and MCF-7 cells, respectively. The highest uptake was found for com-
a
Using a gradient of aqueous 0.1% CF3COOH and methanol as the solvent.
The values in parentheses are for the Re congeners.
b
3. Biological evaluation
To assess the ability of Tc1–Tc4 to target mitochondria we have
performed their in vitro biological evaluation, which comprised cellu-
lar uptake studies in a panel of human tumor cells, including in some
cases the corresponding P-glycoprotein (Pgp)-expressing cell lines,
and the measurement of their uptake in isolated mitochondria. The
plex Tc2 in PC-3 cell line (8.30%
0.63, after 4 h of incubation).
As commented in the introductory part, the cell uptake of lipophilic
and cationic radiotracers, such as 99mTc-Sestamibi can be affected by the
expression of Pgp. Hence, we have checked if expression of Pgp would
affect the cellular uptake of Tc1–Tc4. To have a first insight into this
Fig. 3. In vitro cell uptake of complexes Tc1–Tc4 in PC-3 (A), MCF-7 (B), MCF-7/MDR1 (C), H69 (D) and H69/Lx4 (E) cell lines.

C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
39
aspect, cellular uptake studies were performed in the Pgp-expressing
cell line MCF-7/MDR1. Unlike Tc2, complexes Tc1, Tc3 and Tc4 showed
a decrease of cellular uptake in the MCF-7/MDR1 cell line when com-
pared to the one obtained in the parental cell line MCF-7 (Figs. 3A, B
and 4A). To further evaluate the effect of Pgp-mediated efflux for com-
plexes Tc1, Tc3 and Tc4 additional studies were performed in the small
cell lung carcinoma cell line H69 and in its derivative drug-resistant cell
line H69/Lx4, which presents Pgp overexpression. As can be seen in
Figs. 3D, E and 4B, the cellular uptake of Tc1 is lower in the drug resis-
tant line (H69/Lx4) with values of 14.68%
0.37 and 11.04%
0.47
for H69 and H69/Lx4, respectively, after 1 h of incubation. The other
two tested complexes, Tc3 and Tc4, presented very similar uptake
values in both cell lines. The expression of Pgp on the different cell
lines has been confirmed by western blot analysis (Fig. 5), using a com-
mercially available antibody as described in Experimental section. The
western blot analysis showed no expression of Pgp in the MCF-7, PC-3
and H69 cell lines, in contrary to the MCF-7/MDR1 and H69/Lx4 cell
lines that showed a band between 150 and 225 kDa in agreement
with the molecular weight of Pgp (170 kDa) [27].
Fig. 5. Western blot analysis of Pgp expression in different cancer cell lines.
had a rather similar uptake, particularly the dicationic compounds Tc1
and Tc2 that showed almost the same uptake values. The lowest
mitochondrial uptake was found for Tc3, while Tc4 has shown the
highest mitochondrial uptake. Tc3 and Tc4 are both monocationic and
lipophilic, being however Tc4 more lipophilic due to the presence of a
p-xylene spacer instead of a butylene one. These findings indicate that
for the monocationic complexes, Tc3 and Tc4, the lipophilicity seems
to play a more important role in the mitochondrial uptake of the com-
plexes. On the contrary, for the dicationic complexes, Tc1 and Tc2, the
influence of lipophilicity seems less important being the presence of
the +2 charge the major driving force of their accumulation in the
mitochondria.
In summary, the comparison of cellular uptake of the 99mTc com-
plexes in the pairs of cell lines MCF-7/MCF-7/MDR1 and H69/H69/Lx4
indicates that Tc1 has a greater tendency to suffer Pgp-mediated efflux
compared with the other complexes under study. However, even for
Tc1 the efflux is much less pronounced than that reported for 99mTc-
Sestamibi in the same cell lines [19,27,28].
3.2. Uptake studies in isolated mitochondria
To assess the mitochondrial targeting ability of the phosphonium-
containing complexes Tc1–Tc4, uptake studies were performed in
isolated mitochondria obtained from PC-3. The isolated mitochondria
were incubated with the compounds at different intervals of time, up
to 2 h of incubation. After appropriate separation of the organelles
from the incubation medium, the radioactivity associated with the
mitochondria was measured and the mitochondrial uptake deter-
mined. The results obtained are presented in Fig. 6.
Tc4, which is the complex that presented the highest binding to
this organelle, was selected for further studies to have an insight
into its targeting ability towards tumoral mitochondria versus normal
mitochondria. Hence, uptake studies of Tc4 were performed in parallel
using isolated mitochondria that were collected from PC-3 cells and
from the human cells of non-tumoral origin HEK. As shown if Fig. 7A,
the binding of Tc4 to tumoral mitochondria is almost 2 times higher
than in normal mitochondria. Consistently, Tc4 reached a cellular up-
take plateau that is significantly higher in PC-3 cells than in HEK cells
(Fig. 7B), with an approximately two-fold reduction of uptake in the
non-tumoral cell line. As mentioned in the introduction, several TPP-
containing 64Cu complexes have been evaluated as PET radioprobes
for the targeting of tumor mitochondria. Some of these 64Cu complexes
have been evaluated in isolated tumor cell mitochondria showing
uptake values up to 1.5%, [13] which are lower than those that we
have obtained for Tc4. These findings suggest that Tc4 has an enhanced
ability to be accumulated by the mitochondria of tumor cells.
After 2 h of incubation, the mitochondrial uptake of Tc1–Tc4
ranged between 1.24 and 3.53%, when expressed in percentage of
uptake per million cells. With the exception of Tc3, the other complexes
Fig. 4. In vitro cellular uptake of complexes Tc1–Tc4: in MCF-7 and MCF-7/MDR1 after
4 h incubation (A) and in H69 and H69/Lx4 after 1 h incubation (B).
Fig. 6. Uptake of complexes Tc1–Tc4 in isolated mitochondria obtained from PC-3 cells.

40
C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
Fig. 7. (A) Comparison of the uptake of Tc4 in isolated mitochondria obtained from
PC-3 and HEK cells; (B) comparison of the in vitro cellular uptake of Tc4 in PC-3 and
HEK cell lines.
3.3. Influence of Δψp and Δψm
The influence of Δψp and Δψm on the cellular uptake was evaluated
for Tc2 and Tc4 using PC-3 cells, as these complexes presented the
highest cellular uptake in this cell line and a considerable uptake in
isolated mitochondria. To evaluate this influence, uptake studies were
run under experimental conditions that can reduce or even collapse
Δψm and Δψp.
Cellular uptake studies were conducted by incubation of PC-3 cells
with the complexes in the presence of a buffer solution with increasing
concentrations of K+ (5 mM, 60 mM and 120 mM) for 4 h. For the K+
concentration of 120 mM, the study was also run in the presence of
valinomycin (Fig. 8A). Δψp is generated mainly by the movement of
K+ ions from inside to outside the cell through K+ channels [29,30].
The increase in K+ concentration in the extracellular medium from
5 mM to 120 mM leads to the establishment of a balance between the
concentrations of K+ inside and outside the cell, and consequently to
membrane depolarization [31]. Valinomycin is a K+ ionophore that
increases membrane permeability to K+. Δψp is nearly zero for high
extracellular concentrations of K+ and, under these conditions, the
addition of valinomycin can lead to the collapse of Δψm due to the
uncoupling of electron chain in oxidative phosphorylation [7,32–35].
As shown in Fig. 8A, the increase in K+ concentration from 5 to
120 mM led to an approximately two-fold reduction of the cellular
uptake for both complexes: from 10.94 to 5.64% and from 8.72 to
4.69% for Tc2 and Tc4, respectively. A further reduction of the uptake,
between 60 and 75%, was found when the uptake was measured in
the presence of valinomycin. Altogether, these data suggested that
both Δψp and Δψm are important factors of the cellular uptake of
these complexes. These findings are in agreement with data from pre-
vious studies with TPP derivatives radiolabelled with 18F and 3H [33].
To further investigate the contribution of Δψp and Δψm to the
cellular uptake of Tc2 and Tc4, the studies were also performed in the
presence of the ionophores nigericin and ouabain (Fig. 8B) and in the
Fig. 8. Effect of alteration in membrane potentials on cellular uptake of complexes Tc2 and
Tc4 in PC-3 cells. Depolarization of Δψm and Δψp: A using increasing concentrations of
K+ (5, 60, 120 mM) and in the presence of valinomycin (Val, 1 μg/mL); B in the presence
of nigericin (5 μg/mL) and nigericin plus ouabain (100 μM); and C in the presence of
increasing concentrations of CCCP (5 and 10 μM).
presence of CCCP (carbonyl cyanide m-chlorophenyl hydrazone)
(Fig. 8C). In these experiments, the cells underwent a prior incubation
with Tc2 and Tc4 followed by incubation with the ionophores or with
the inhibitor. Nigericin mediates electroneutral K+/H+ exchange and
is known to collapse the pH gradient across mitochondrial inner mem-
branes, thereby producing a secondary hyperpolarization of Δψm [36].
Nigericin can also induce the hyperpolarization of the plasma mem-
brane but this can be avoided by the presence of ouabain, which is a
specific inhibitor of the plasma membrane Na+/K+ ATPase [32,37].
Finally, CCCP is an inhibitor of oxidative phosphorylation, uncoupling
the proton gradient across the inner mitochondrial membrane and
resulting in the collapse of Δψm [38].
The addition of nigericin induced an increase in the cellular uptake
of Tc2 and Tc4, between 30 and 40%, which most probably reflects the
combined effects of mitochondrial hyperpolarization and the hyper-
polarization of the plasma membrane. As shown in Fig. 8B, the further

C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
41
addition of ouabain reduced this increase, but the uptake values
were still higher than those obtained in the control experiment
(i.e. without addition of nigericin and ouabain) with values of 7.03
vs 6.02% (Tc2) and 5.39 vs 4.78% (Tc4), respectively. This trend also
indicates that Δψm may play a role in the cellular uptake of Tc2 and
Tc4. The addition of CCCP at a concentration of 10 μM led to a
decrease in the uptake, with values changing from 6.02 to 3.55% (Tc2)
and from 4.78 to 3.08% (Tc4) (Fig. 7C), which is also consistent with
the contribution of Δψm in the cellular uptake of the complexes.
60 F254 plates, and Instant Thin-layer chromatography (ITLC) on PALL
ITLC SG strips. Column chromatography was performed with silica gel
60 (Merck). HPLC analysis of the Re and 99mTc complexes was performed
on a Perkin-Elmer LC pump 200 coupled to a LC 290 tunable UV–vis
detector and to a Berthold LB-507A radiometric detector, using an
analytical Macherey-Nagel C18 reversed-phase column (Nucleosil
100–10, 250 × 3 mm) with a flow rate of 1 mL min−1. HPLC purifica-
tions were performed using a semi-preparative Macherey–Nagel C18
reversed-phase column (Nucleosil 100–7, 250 × 8 mm) with a flow
rate of 2.0 mL min−1 or a using a preparative Waters μ Bondapak C18
column (150 × 19 mm) at a flow rate of 5.0 mL min−1, UV detection,
254 nm. Gradient elution was used both for analytical and semi-
preparative HPLC, with the solvent systems: A — aqueous 0.1% CF3COOH
solution, B — MeOH. The gradient systems used are indicated below for
each compound.
4. Conclusions
We succeeded in the synthesis of the first examples of TPP-
containing organometallic Re(I)/99mTc(I) complexes. The synthesized
complexes presented a dicationic (Re1/Tc1, Re2/Tc2) or monocationic
character (Re3/Tc3, Re4/Tc4). Their lipophilicity was readily tuned by
the use of different spacers (butylene vs p-xylene) to link the TPP
pharmacophore to the central amine of the corresponding tridentate
chelators.
5.1.1. (4-((2-((Tert-butoxycarbonyl)amino)ethyl)(2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethyl)amino)butyl)triphenylphosphonium bromide, 2
To a solution of tert-butyl (2-((2-(3,5-dimethyl-1H-pyrazol-1-yl)
ethyl)amino)ethyl)carbamate (1) (668 mg, 1.95 mmol) and (4-
bromobutyl)triphenylphosphonium bromide (934 mg, 1.95 mmol) in
anhydrous DMF (25 mL) was added triethylamine (density =
0.726 g/mL) (546 μL, 3.92 mmol). The reaction mixture was left to stir
at room temperature for 72 h. After removal of the solvent, CH2Cl2
and H2O were added to the residue. The organic phase was separated,
dried over MgSO4, filtered and after removal of the solvent the product
was further purified by silica gel column chromatography, using
CH2Cl2/MeOH (95/5) as eluent. The final compound (64%, 850 mg)
was recovered from the collected fractions as a yellow oil, after removal
of the solvent under reduced pressure.
The in vitro evaluation of Tc1–Tc4 has shown that these complexes
accumulate in different types of tumor cells, and has also proved that
their cell uptake values are not much affected by the expression of
Pgp. The mitochondria seem to be a relevant intracellular target of
Tc1–Tc4, as pointed out by the results obtained with isolated mitochon-
dria. For Tc1 and Tc3, presenting a butylenic spacer to link the TPP
pharmacophore, there is an enhancement of the mitochondrial uptake
for the dicationic complex (Tc1) compared with the monocationic
counterpart (Tc3), as predicted by the Nernst equation. However,
such enhancement was not observed for the pair of complexes Tc2
and Tc4, showing that the lipophilicity is also a very important factor
of the ability of these complexes to cross the mitochondria membrane.
For the complex with the highest mitochondrial uptake, Tc4, the in
vitro studies suggested the accumulation in mitochondria of tumor
cells by diffusion, in favor of the electrochemical gradient and showing
a strong dependency on the mitochondrial and plasma membrane
potentials. Moreover, Tc4 has shown a higher affinity towards tumor
mitochondria than towards normal mitochondria, as well as a higher
uptake in human tumor cells if compared with normal cells from
human origin. Altogether, these results indicate that the new TPP-
containing 99mTc(I) tricarbonyl complexes reported in this manuscript
are promising platforms to design radioactive metalloprobes targeted
to the mitochondria for in vivo detection of tumor tissues.
Rf (SiO2, CH2Cl2/MeOH (90/10)) = 0.42; 1H NMR (CDCl3): δH 1.23
(9H, s, BOC), 1.44 (2H, m, CH2), 1.61 (2H, m, CH2), 1.96 (3H, s,
CH3-pz), 2.03 (3H, s, CH3-pz), 2.35 (4H, m, 2 CH2), 2.64 (2H, m,
CH2), 2.86 (2H, m, CH2), 3.52 (2H, m, CH2), 3.79 (2H, m, CH2), 5.15
(1H, s, H4-pz), 7.55–7.73 (15H, m, PPh3); 13C NMR (CDCl3): δc 11.04
(CH3-pz), 13.07 (CH3-pz), 20.00 (d, JP-C = 13 Hz, CH2), 22.82 (d,
JP-C = 46 Hz, CH2), 26.70 (d, JP-C = 18 Hz, CH2), 28.07 (CH3-BOC),
38.14 (CH2), 44.33 (CH2), 46.77 (CH2), 53.25 (CH2), 54.15 (CH2),
78.31 (Cquaternary-BOC), 104.35 (C4-pz), 117.51 (d, JP-C = 85 Hz, PPh3),
130.18 (d, JP-C = 12 Hz, PPh3), 133.26 (d, JP-C = 10 Hz, PPh3), 135.34
(d, JP-C = 3.7 Hz, PPh3), 138.82 (C3/5-pz), 146.62 (C3/5-pz), 155.80
(C_O); 31P NMR (CDCl3) δP 23.88; ESI/MS (+) (m/z): 599.4 M+
,
calcd for C36H48N4O2P = 599.4 [M+]; Anal. Calc. for C36H48N4O2PBr:
C, 63.60; H, 7.12; N, 8.25, Found: C, 63.58; H, 6.86; N, 8.35.
5. Experimental section
5.1. Chemistry
5.1.2. (4-(((2-((Tert-butoxycarbonyl)amino)ethyl)(2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethyl)amino)methyl)benzyl)triphenylphosphonium bromide, 3
Compound 3 (47%, 858 mg) was prepared and purified as above
described for 2, starting from 709 mg (2.49 mmol) of compound
1 and 1.310 g (2.49 mmol) of (4-(bromomethyl)benzyl)triphenyl-
phosphonium bromide.
Unless otherwise stated, the synthesis of the ligands and com-
plexes was carried out under a nitrogen atmosphere, using standard
Schlenk techniques and dry solvents; the work-up procedures were
performed under air. The compounds, tert-butyl (2-((2-(3,5-dimethyl-
1H-pyrazol-1-yl)ethyl)amino)ethyl)carbamate (1) [39], ethyl 2-((2-
(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)amino)acetate (4) [24] and (4-
(bromomethyl)benzyl)triphenylphosphonium bromide [13] were
prepared according to published methods. The starting material fac-
[Re(H2O)3(CO)3]Br was synthesized by the literature method [40] Na
Rf (SiO2, CH2Cl2/MeOH (90/10)) = 0.56; 1H NMR (CDCl3): δH 1.21
(9H, s, BOC), 1.82 (3H, s, CH3-pz), 1.97 (3H, s, CH3-pz), 2.35 (2H, t,
CH2), 2.52 (2H, t, CH2), 2.91 (2H, t, CH2), 3.32 (2H, s, CH2), 3.75 (2H,
t, CH2), 5.02 (2H, d, CH2), 5.42 (1H, bs, NH), 5.51 (1H, s, H4-pz),
6.67–6.77 (4H, m, Ph), 7.43–7.61 (15H, m, PPh3); 13C NMR (CDCl3):
δc 10.48 (CH3-pz), 12.95 (CH3-pz), 28.01 (CH3-BOC), 30.03 (d,
JP-C = 47 Hz, CH2), 37.94 (CH2), 46.15 (CH2), 52.71 (CH2), 53.18
(CH2), 57.62 (CH2), 78.23 (Cquaternary-BOC), 104.52 (C4-pz), 117.12
(d, JP-C = 86 Hz, PPh3), 125.10 (d, JP-C = 8.3 Hz, CPh), 128.60 (d,
[
99mTcO4] was eluted from a commercial 99mo/99mTc generator, using
0.9% saline. 1H, 13C and 31P NMR spectra were recorded on a Varian
Unity 300 MHz spectrometer. 1H, 13C and 31P chemical shifts are given
in ppm; 1H and 13C chemical shifts were referenced with the residual sol-
vent resonances relative to SiMe4 and the 31P chemical shifts were
referenced with an external 85% H3PO4 solution. IR spectra were
recorded as KBr pellets on a Bruker, Tensor 27 spectrometer. ESI-MS
was performed at Instituto Tecnológico e Nuclear (ITN) on a quadrupole
ion trap mass spectrometry (QITMS) instrument in positive ion mode.
Thin-layer chromatography (TLC) was performed on Merck silica gel
JP-C = 2.3 Hz, Cbz), 129.73 (d, JP-C = 12 Hz, PPh3), 130.73 (d, JP-C
=
5 Hz, CPh), 133.74 (d, JP-C = 10 Hz, PPh3), 134.61 (d, JP-C = 2.3 Hz,
PPh3), 138.28 (C3/5-pz), 139.03 (d, JP-C = 3.8 Hz, Cbz), 146.54 (C3/5-pz),
155.76 (C_O); 31P NMR (CDCl3) δP 23.67; ESI/MS (+) (m/z):
647.4 M+ calcd for C40H48N4O2P = 647.4 [M+]; Anal. Calc. for
,

42
C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
C40H48N4O2PBr: C, 66.00; H, 6.65; N, 7.70, Found: C, 65.92; H,1 5.78; N,
7.57.
(CH2), 47.32 (CH2), 51.94 (CH2), 54.15 (CH2), 54.37 (CH2), 106.15
(C4-pz)), 115.54 (CTFA), 119.87 (d, JP-C = 87 Hz, PPh3), 131.55 (d,
JP-C = 13 Hz, PPh3), 134.81 (d, JP-C = 10 Hz, PPh3), 136.28 (d,
JP-C = 3.4 Hz, PPh3), 141.27 (C3/5-pz)), 148.49 (C3/5-pz), 161.50
5.1.3 (4-((2-(3,5-Dimethyl-1H-pyrazol-1-yl)ethyl)(2-ethoxy-2-oxoethyl)
amino)butyl)triphenylphosphonium bromide, 5
(CTFA); 31P NMR (CD3OD) δP 24.67; ESI/MS (+) (m/z): 499.4 M+
,
calcd for C31H40N4P = 499.3 [M+]; Anal. Calc. for C33H40N4PF3O2.2
CF3COOH: C, 52.86; H, 5.04; N, 6.66, Found: C, 52.81; H1, 5.70; N, 6.93.
Compound 5 (44%, 1.149 g) was prepared and purified as above
described for 2, starting from 957 mg (4.25 mmol) of ethyl 2-((2-
(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)amino)acetate (4) and 2.032 g
(4.25 mmol) of (4-bromobutyl)triphenylphosphonium bromide.
Rf (SiO2, CH2Cl2/MeOH (90/10)) = 0.47; 1H NMR (CDCl3): δH 1.09
(3H, t, CH3), 1.53 (2H, m, CH2), 1.66 (2H, m, CH2), 1.99 (3H, s,
CH3-pz), 2.00 (3H, s, CH3-pz), 2.50 (2H, t, CH2), 2.78 (2H, t, CH2),
3.12 (2H, s, CH2), 3.58 (2H, m, CH2), 3.83 (2H, t, CH2), 3.95 (2H, q,
CH2), 5.49 (1H, s, H4-pz), 7.56–7.75 (15H, m, PPh3); 13C NMR
(CDCl3): δc 12.87 (CH3-pz), 15.16 (CH3-pz), 15.93 (CH3), 21.64 (d,
5.1.6. (4-(((2-Aminoethyl)(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)amino)
methyl)benzyl)triphenylphosphonium trifluoroacetate, L2
To a solution of 3 (858 mg, 1.18 mmol) in CH2Cl2 (5 mL) was added
trifluoroacetic acid (density = 1.489 g/mL) (3.00 mL, 39.20 mmol),
and the mixture was stirred overnight at room temperature. The
solvent was removed under vacuum yielding compound L2 (1.157 g,
99%) as a yellow oil, after removal of the solvent under vacuum.
1H NMR (CD3OD): δH 2.25 (3H, s, CH3-pz); 2.28 (3H, s, CH3-pz);
3.30 (6H, m, 3 CH2); 4.18 (2H, t, CH2); 4.55 (2H, t, CH2); 4.86 (2H,
d, CH2); 6.16 (1H, s, H4-pz); 7.02–7.34 (4H, m, Ph); 7.57–7.86 (15H,
m, PPh3); 13C NMR (CD3OD): δc 10.91 (CH3-pz), 11.57 (CH3-pz),
30.79 (d, JP-C = 49 Hz, CH2), 36.57 (CH2), 45.14 (CH2), 51.53 (CH2),
52.80 (CH2), 57.62 (CH2), 108.57 (C4-pz), 118.51 (d, JP-C = 87 Hz,
JP-C = 4.5 Hz, CH2), 23.69 (d, JP-C = 50 Hz, CH2), 29.34 (d, JP-C
=
17 Hz, CH2), 48.82 (CH2), 55.10 (CH2), 55.35 (CH2), 57.22 (CH2),
62.14 (CH2), 106.44 (C4-pz)), 119.93 (d, JP-C = 86 Hz, PPh3), 132.22
(d, JP-C = 15 Hz, PPh3), 135.38 (d, JP-C = 9.8 Hz, PPh3), 136.76 (d,
JP-C = 2.3 Hz, PPh3), 140.92 (C3/5-pz), 148.83 (C3/5-pz)), 173.10
(C_O); 31P NMR (CDCl3) δP 23.89; ESI/MS (+) (m/z): 542.3 M+
,
calcd for C33H41N3O2P = 542.3 [M+]; Anal. Calc. for C33H41N3O2PBr:
C, 63.65; H1, 6.64; N, 6.75, Found: C, 63.58; H1, 5.97; N, 6.89.
PPh3), 118.59 (CTFA), 129.72 (d, JP-C = 8.3 Hz, Cbz), 131.32 (d, JP-C
=
13 Hz, PPh3), 132.03 (d, JP-C = 2.3 Hz, Cbz), 132.78 (d, JP-C = 10 Hz,
PPh3), 134.61 (d, JP-C = 5 Hz, Cbz), 135.15 (d, JP-C = 2.3 Hz, PPh3),
136.20 (d, JP-C = 3.8 Hz, Cbz), 136.49 (C3/5-pz)), 148.01 (C3/5-pz)),
161.14 (CTFA); 31P NMR (CD3OD) δP 23.76; ESI/MS (+) (m/z):
5.1.4. (4-(((2-(3,5-Dimethyl-1H-pyrazol-1-yl)ethyl)(2-ethoxy-2-oxoethyl)
amino)methyl)benzyl)triphenylphosphonium bromide, 6
Compound 6 (22%, 524 mg) was prepared and purified as above
described for 2, starting from 815 mg (3.62 mmol) of compound 4
and 1.901 g (3.62 mmol) of (4-(bromomethyl)benzyl)triphenyl-
phosphonium bromide.
647.4 M+ calcd for C40H48N4O2P = 647.4 [M+]; Anal. Calc. for
,
C42H48N4PF3O2.2 CF3COOH: C, 55.87; H, 5.10; N, 5.67, Found: C, 55.91;
H, 5.52; N 5.47.
Rf (SiO2, CH2Cl2/MeOH (90/10)) = 0.46; 1H NMR (CDCl3): δH 1.05
(3H, t, CH3); 1.96 (6H, s, 2 CH3-pz); 2.82 (2H, t, CH2); 3.03 (2H, s,
CH2); 3.52 (2H, s, CH2); 3.77 (2H, t, CH2); 3.91 (2H, q, CH2); 5.07
(2H, d, CH2); 5.55 (1H, s, H4-pz); 6.82 (4H. s, Ph); 7.42–7.62 (15H,
m, PPh3); 13C NMR (CDCl3): δc 11.23 (CH3), 13.69 (CH3-pz), 14.43
(CH3-pz), 30.64 (d, JP-C = 47 Hz, CH2), 47.57 (CH2), 53.84 (CH2),
54.60 (CH2), 58.24 (CH2), 60.51 (CH2), 105.05 (C4-pz)), 117.75 (d,
5.1.7.
(2-((2-(3,5-Dimethyl-H-pyrazol-1-yl)ethyl)(4-(triphenylphos-
phonio)butyl)amino)acetate, L3H
Compound 4 (323 mg, 0.52 mmol) was dissolved in 4 M HCl
(12 mL), and the resulting solution was refluxed overnight. After
cooling to room temperature, the solution was neutralized to pH 7
and the solvent removed under vacuum. The residue was redissolved
in the minimum amount of water and applied on a preconditioned
C18 cartridge (Sep-Pak, Waters). The cartridge was eluted with dis-
tilled water to remove the salts and then with methanol to recover
the title compound. After removal of the solvent from the methanolic
fractions, L3H (158 mg, 50%) was recovered as a yellow oil.
IR υmax/cm−1: 1583 (C_O); 1H NMR (CD3OD): δH 1.66 (4H, m, 2
CH2); 2.10 (3H, s, CH3-pz); 2.18 (3H, s, CH3-pz); 2.62 (2H, m, CH2);
2.85 (2H, t, CH2); 3.13 (2H, c, CH2); 3.51 (2H, m, CH2); 4.02 (2H, t,
CH2); 5.67 (1H, s, H4-pz); 7.74–7.91 (15H, m, PPh3); 13C NMR
(CD3OD): δc 11.19 (CH3-pz), 13.33 (CH3-pz), 21.12 (d, JP-C = 4.5 Hz,
CH2), 22.24 (d, JP-C = 51 Hz, CH2), 28.73 (d, JP-C = 17 Hz, CH2),
47.60 (CH2), 54.49 (CH2), 54.97 (CH2), 59.93 (CH2), 105.76 (C4-pz)),
120.00 (d, JP-C = 87 Hz, PPh3), 131.50 (d, JP-C = 12 Hz, PPh3),
134.85 (d, JP-C = 10 Hz, PPh3), 136.13 (d, JP-C = 3.0 Hz, PPh3), 141.28
(C3/5-pz)), 148.30 (C3/5-pz)), 178.86 (C_O); 31P NMR (CD3OD) δP
25.15; ESI/MS (+) (m/z): 514.3 M+, calcd for C31H37N3O2P = 514.3;
Anal. Calc. for C31H36N3PO2: C, 72.49; H, 7.06; N, 8.18, Found: C, 72.36;
H, 6.91; N, 8.05.
JP-C = 86 Hz, PPh3), 126.10 (d, JP-C = 8.3 Hz, CPh), 129.37 (d, JP-C
3.0 Hz, CPh), 130.33 (d, JP-C = 19 Hz, PPh3), 131.53 (d, JP-C = 6.0 Hz,
Ph), 134.38 (d, JP-C = 9.3 Hz, PPh3), 135.25 (d, JP-C = 3.0 Hz, PPh3),
=
C
139.20 (C3/5-pz)), 139.43 (d, JP-C = 3.8 Hz, CPh), 147.35 (C3/5-pz)),
171.26 (C_O); 31P NMR (CDCl3) δP 24.21; ESI/MS (+) (m/z):
590.3 M+ calcd for C37H41N3O2P = 590.3 [M+]; Anal. Calc. for
,
C37H41N3O2PBr: C, 66.25; H, 6.17; N, 6.27, Found: C, 66.15; H, 5.89; N,
6.59.
5.1.5. (4-((2-Aminoethyl)(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)amino)
butyl)triphenylphosphonium trifluoroacetate, L1
To a solution of 2 (850 mg, 1.25 mmol) in CH2Cl2 (5 mL) was added
trifluoroacetic acid (density = 1.489 g/mL) (1.62 mL, 21.10 mmol),
and the mixture was stirred overnight at room temperature. The sol-
vent was removed under vacuum and the crude product was purified
by RP-HPLC on a preparative Waters μ Bondapack. Method: 0–3 min,
75% A; 3–23 min, 75%–50% A; 23–33 min, 50%; 33–43 min 50–0% A;
43–58 min 0% A (tR = 16.35 min). Compound L1 (458 mg, 44%) was
recovered from the collected fractions as a transparent oil, after removal
of the solvent under vacuum.
5.1.8. 2-((2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)(4-((triphenylphos-
phonio)methyl)benzyl)amino)acetate, L4H
Compound L4H (68%, 302 mg) was prepared as above described
for L3H, starting from 432 mg (0.66 mmol) of 6.
1H NMR (CD3OD): δH 1.62 (4H, m, 2 CH2); 2.11 (3H, s, CH3-pz);
2.21 (3H, s, CH3-pz); 2.51 (2H, t, CH2); 2.80 (2H, m, CH2); 2.83 (2H,
t, CH2); 2.95 (2H, t, CH2); 3.37 (2H, m, CH2); 4.03 (2H, m, CH2);
5.79 (1H, s, H4-pz); 7.72–7.92 (15H, m, PPh3); 13C NMR (CD3OD):
δc 10.94 (CH3-pz), 13.21 (CH3-pz), 21.27 (d, JP-C = 12 Hz, CH2),
22.56 (d, JP-C = 51 Hz, CH2), 28.70 (d, JP-C = 17 Hz, CH2), 38.58
IR υmax/cm−1: 1586 (C_O); 1H NMR (CD3OD): δH 2.05 (3H, s,
CH3-pz); 2.18 (3H, s, CH3-pz); 3.18 (2H, t, CH2); 3.42 (2H, s, CH2);
3.98 (2H, s, CH2); 4.55 (2H, t, CH2); 4.96 (2H, d, CH2); 5.77 (1H, s,
H4-pz); 6.97 (2H, m, Ph); 7.22 (2H, m, Ph); 7.61–7.91 (15H, m,
PPh3); 13C NMR (CD3OD): δc 10.93 (CH3-pz), 13.28 (CH3-pz), 30.33
(d, JP-C = 47 Hz, CH2), 45.84 (CH2), 54.80 (CH2), 56.58 (CH2), 59.34
(CH2), 106.17 (C4-pz)), 119.11 (d, JP-C = 86 Hz, PPh3), 128.72 (d,
1
Although the H analysis data for this compound are somewhat unsatisfactory, the
ESI-MS results and 1H, 13C and 31P NMR data clearly support the proposed formulation.

C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
43
JP-C = 8.8 Hz, CPh), 131.35 (d, JP-C = 18 Hz, PPh3), 132.35 (d, JP-C
3.3 Hz, CPh), 132.78 (d, JP-C = 9.0 Hz, PPh3), 134.52 (d, JP-C = 6.0 Hz,
=
(CH2); 63.86 (CH2); 66.34 (CH2); 108.89 (C4-pz)); 115.37 (CTFA);
119.67 (d, JP-C = 87 Hz, PPh3); 131.62 (d, JP-C = 12 Hz, PPh3); 134.84
(d, JP-C = 11 Hz, PPh3); 136.42 (d, JP-C = 3.0 Hz, PPh3); 145.07
(C3/5-pz)); 155.22 (C3/5-pz)); 161.37 (CTFA); 181.54 (C_O); 195.09
(C`O); 196.38 (C`O); 196.85 (C`O); 31P NMR (CD3OD) δP 24.56;
ESI/MS (+) (m/z): 784.1 M+, calcd for ReC34H36N3O5P = 784.2; Anal.
Calc. for ReC36H36N3O7PF3.3CF3COOH: C, 40.72; H, 3.17; N, 3.39,
Found: C, 40.89; H, 2.94; N 3.55.
C
Ph), 135.41 (d, JP-C = 14 Hz, PPh3), 136.45 (d, JP-C = 3.2 Hz, CPh),
141.49 (C3/5-pz)), 148.90 (C3/5-pz)), 174.19 (C_O); 31P NMR (CD3OD)
δP 23.79; ESI/MS (+) (m/z): 562.3 M+, calcd for C35H37N3O2P = 562.3;
Anal. Calc. for C35H36N3PO2: C, 74.71; H, 6.63; N, 7.47, Found: C, 74.52;
H, 6.25; N, 7.37.
5.1.9. General procedure for the synthesis of the Re complexes, Re1–Re4
fac-[Re(H2O)3(CO)3]Br was reacted with equimolar amounts of
L1–L4H in refluxing methanol (Re1–Re2) or H2O (Re3–Re4) for
18 h. After this time, the solvent was removed under vacuum and the
complexes were purified by RP-HPLC, except Re1 that was purified by
washing with adequate solvents.
5.1.9.4. fac-[Re(CO)3(κ3-L4)](CF3COO), Re4. Re4 (27%, 30 mg) was
purified by RP-HPLC. Method: 0–3 min. 75% A, 3–3.1 min. 75%–50%
A, 3.1–9.0 min. 50% A, 9.0–9.1 min 50–35% A, 9.1–30 min 35-0% A,
30–35 min 0% A.
IR υmax/cm−1: 1647 (C_O) 1904, 2020 (C`O); 1H NMR (CD3OD):
δH 2.25 (1H, m, CH2), 2.31 (3H, s, CH3-pz), 2.48 (3H, s, CH3-pz), 3.04
(1H, d, CH2), 3.25 (1H, m, CH2), 3.94 (1H, d, CH2), 4.41 (2H, m,
CH2), 4.62 (2H, m, CH2), 4.97 (2H, m, CH2), 6.13 (1H, s, H4-pz), 7.15
(2H, d, Ph), 7.48 (2H, d, Ph), 7.63–7.92 (15H, m, PPh3); 13C NMR
(CD3OD): δc 10.20 (CH3-pz), 14.87 (CH3-pz), 29.02 (d, JP-C = 48 Hz,
CH2), 45.05 (CH2), 55,19 (CH2), 62.73 (CH2), 67.68 (CH2), 107.61
(C4-pz), 117.86 (d, JP-C = 87 Hz, PPh3), 119.03 (CTFA), 128.83 (d,
5.1.9.1. fac-[Re(CO)3(κ3-L1)]Br2, Re1. Re1 (59%, 38 mg) was purified
by washing with n-hexane and H2O.
IR υmax/cm−1: 1897, 2025 (C`O); 1H NMR (CD3OD): δH 1.77 (2H,
m, CH2), 2.03 (1H, m, CH2), 2.27 (1H, m, CH2), 2.37 (3H, s, CH3-pz),
2.43 (3H, s, CH3-pz), 2.66 (1H, m, CH2), 2.74 (1H, m, CH2), 2.86 (2H,
m, CH2), 3.16 (1H, m, CH2), 3.44 (1H, m, CH2), 3.62 (4H, m,
CH2 + CH2 + CH2), 4.04 (1H, m, NH2), 4.22 (1H, m, CH2), 4.52 (1H,
m, CH2), 5.46 (1H, m, NH2), 6.19 (1H, s, H4-pz), 7.74–7.93 (15H, m,
PPh3); 13C NMR (CD3OD): δc 11.63 (CH3-pz), 16.05 (CH3-pz), 21.17
(CH2), 22.81 (d, JP-C = 52 Hz, CH2), 26.48 (d, JP-C = 17 Hz, CH2),
43.66 (CH2), 48.45 (CH2), 54.09 (CH2), 62.67 (CH2), 67.04 (CH2),
JP-C = 12 Hz, Ph), 130.30 (d, JP-C = 14 Hz, PPh3), 131.43 (d, JP-C
=
4.5 Hz, Ph), 131.92 (d, JP-C = 10 Hz, PPh3), 133.10 (d, JP-C = 5.8 Hz,
Ph), 134.19 (d, JP-C = 10 Hz, PPh3), 135.43 (d, JP-C = 2.2 Hz, Ph),
144.16 (C3/5-pz)), 154.35 (C3/5-pz)), 160.70 (CTFA), 180.00 (C_O),
193.90 (C`O), 194.89 (C`O), 195.63 (C`O); 31P NMR (CD3OD) δP
24.14; ESI/MS (+) (m/z): 832.2 M+, calcd for ReC38H36N3O5P =
832.2; Anal. Calc. for ReC40H36N3O7PF3.2CF3COOH: C, 45.06; H, 3.27;
N, 3.58, Found: C, 45.16; H 3.56; N, 3.37.
109.16 (C4-pz)), 119.65 (d, JP-C = 81 Hz, PPh3), 131.55 (d, JP-C
=
12 Hz, PPh3), 134.87 (d, JP-C = 10 Hz, PPh3), 136.29 (d, JP-C = 3.0 Hz,
PPh3), 145.29 (C3/5-pz), 155.00 (C3/5-pz), 193.66 (C`O), 194.73
(C`O), 195.15 (C`O); 31P NMR (CD3OD) δP 24.64; ESI/MS (+)
(m/z): 385.2 M2+, calcd for ReC34H40N4O3PBr2 = 770.3; Anal. Calc.
for ReC34H40N4O3PBr2: C, 43.93; H, 4.34; N, 6.03, Found: C, 43.79; H,
4.45; N 6.37.
5.2. Synthesis of 99mTc(I) complexes, Tc1–Tc4
5.2.1. General method
In a nitrogen-purged glass vial, 40 μL of a 4.4 × 10−3 M aqueous
solution of L1–L4H was added to 400 μL of a solution of the organo-
metallic precursor fac-[99mTc(H2O)3(CO)3]+ (1–2 mCi) in saline at
pH 7.4 (Tc1–Tc2) or at pH 5.5 (Tc3–Tc4). The reaction mixture was
heated to 100 °C for 30 min, cooled to room temperature and ana-
lyzed by RP-HPLC. Method: 0–3 min, 100% A; 3–3.1 min, 100%–75%
A; 3.1–9 min, 75% A; 9–9.1 min 75%–66% A; 9,1–20 min, 66%–0% A;
20–25 min, 0% A; 25–25.1 min, 0%–100% A; 25.1–30 min, 100% A.
5.1.9.2. fac-[Re(CO)3(κ3-L2)](CF3COO)2, Re2. Re2 (45%, 62 mg) was
purified by RP-HPLC. Method: 0–3 min, 75% A; 3–28 min, 75%–50%
A; 28–33 min, 50%; 33–48 min 50–0% A; 48–58 min 0% A.
IR υmax/cm−1: 1899, 2027 (C`O); 1H NMR (CD3OD): δH 2.36 (3H.
s, CH3-pz); 2.45 (3H, s, CH3-pz); 2.52 (3H, m, CH2 + CH2 + CH2);
2.90 (1H, d, CH2); 3.02 (2H, m, CH2 + CH2); 4.23 (1H, m, CH2); 4.35
(1H, m, CH2); 4.53 (1H, m, CH2); 4.61 (1H, m, CH2); 4.80 (2H, d,
CH2); 5.61 (2H, bs, NH2); 6.19 (1H, s, H4-pz); 7.12 (2H, d, Ph); 7.41
(2H, d, Ph); 7.67–7.92 (15H, m, PPh3); 13C NMR (CD3OD): δc 11.54
(CH3-pz), 16.17 (CH3-pz), 30.15 (d, JP-C = 48 Hz, CH2), 43.28 (CH2),
47.17 (CH2), 51.90 (CH2), 62.13 (CH2), 69.47 (CH2), 109.09 (C4-pz),
5.3. Distribution coefficient measurements
The log Do/w values of complexes Tc1–Tc4 were determined by the
“shake flask” method under physiological conditions (n-octanol–
0.1 M PBS, pH 7.4) [41].
118.97 (d, JP-C = 87 Hz, PPh3), 118.53 (CTFA), 130.63 (d, JP-C
8.3 Hz, CPh), 131.43 (d, JP-C = 13 Hz, PPh3), 132.59 (d, JP-C = 5.3 Hz,
Ph), 133.35 (d, JP-C = 10 Hz, PPh3), 134.58 (d, JP-C = 3.0 Hz, CPh),
=
C
135.34 (d, JP-C = 2.3 Hz, PPh3), 136.57 (d, JP-C = 3.8 Hz, CPh), 145.62
(C3/5-pz), 155.50 (C3/5-pz)), 161.70 (CTFA), 193.72 (C`O), 194.60
(C`O), 195.21 (C`O); 31P NMR (CD3OD) δP 24.05; ESI/MS (+)
(m/z): 409.1 M2+, calcd for ReC38H40N4O3P = 818.3; Anal. Calc. for
ReC42H40N4O7PF6.CF3COOH: C, 45.64; H, 3.57; N 4.84, Found: C, 45.66;
H, 3.05; N, 4.77.
5.4. Cell culture
B16-F1 murine melanotic melanoma, HEK human embryonic
kidney (ECACC, UK), MCF-7 human breast cancer (ATCC, Spain) and
MCF-7/MDR1 (kindly provided by Professor David Piwnica-Worms,
Washington University School of Medicine, St Louis, Missouri, USA) [42],
were grown in DMEM (Dulbecco's modified eagle's medium) containing
GlutaMax I, while PC-3 human prostate cancer (ATCC, Spain), H69
and H69/Lx4 small cell lung human carcinoma (ECACC, UK) were
grown in RPMI 1640 medium. The media were supplemented with
10% heat-inactivated fetal bovine serum and 1% penicillin/strepto-
mycin antibiotic solution (all from Invitrogen). The MCF-7/MDR1
and H69/Lx4 media were supplemented with geneticin 1 mg/mL
(Invitrogen) and doxorubicin 0.4 μg/mL (Sigma-Aldrich), respectively.
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. For adherent cell lines (B16-F1, A375, PC-3, MCF-7 and MCF-7/
MDR1) the cells were adherent in monolayers and, when confluent,
5.1.9.3. fac-[Re(CO)3(κ3-L3)](CF3COO), Re3. Re3 (45%, 62 mg) was
purified by RP-HPLC. Method: 0–3 min, 75% A; 3–3.1 min, 75%–50%
A; 3.1–9.0 min, 50%; 9.0–9.1 min 50–35% A; 9.1–30 min 35–0% A;
30–35 min 0% A.
IR υmax/cm−1: 1644 (C_O) 1981, 2027 (C`O); 1H NMR (CD3OD):
δH 1.76 (2H, m, CH2); 2.01 (2H, m, CH2); 2.32 (3H, s, CH3-pz); 2.44
(3H, s, CH3-pz); 2.56 (1H, m, CH2); 3.23 (1H, d, CH2); 3.45 (5H, m,
CH2 + CH2 + CH2); 3.64 (1H, d, CH2); 4.34 (2H, m, CH2); 6.13 (1H,
s, H4-pz); 7.73-7.93 (15H, m, PPh3); 13C NMR (CD3OD): δc 11.34
(CH3-pz); 15.72 (CH3-pz); 21.05 (d, JP-C = 3.0 Hz, CH2); 22.75 (d,
JP-C = 52 Hz, CH2); 26.28 (d, JP-C = 17 Hz, CH2); 46.74 (CH2); 56.84

44
C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
were harvested from the cell culture flasks with trypsin–EDTA
(Invitrogen) and seeded in new culture flasks.
For the suspension cell lines, H69 and H69/Lx4, the cells were
transferred from culture flasks to plastic tubes, centrifuged for
5 min at 850 g, and the cell pellet resuspended in fresh medium
and seeded in new culture flasks.
fraction) and pellet corresponded to the mitochondrial fraction. The
pellet was washed again with 300 μL of Reagent C and centrifuged to
12,000 g for 5 min. The pellet containing the isolated mitochondria
was kept on ice before being used in studies of mitochondrial uptake.
5.7. Mitochondrial uptake
5.5. Cellular uptake studies
The mitochondrial pellet isolated from approximately 10–15 ×
106 cells was resuspended in 2 mL of culture medium. The mitochon-
drial suspension was incubated with the complexes under study for
15 min, 30 min, 1 h and2 h and 3 h in constant agitation at 37 °C.
After the incubation period aliquot samples of 200 μL (in duplicate)
were collected to microfuge tubes containing 500 μL of cold PBS.
The samples were centrifuged for 3 min at 9400 g (Centrifuge
5417R, Eppendorf). After discarding the supernatant, the pellet was
washed again with 500 μL of cold PBS, and samples centrifuged
(10 min, 9400 g). The activity of the cell pellet was measured in
gamma counter. The % of mitochondrial uptake was calculated as
described for cellular uptake.
Prior to the cellular uptake assays of the 99mTc complexes (Tc1–
Tc4), their stability in the cell culture media was evaluated by incu-
bating the complexes with the media at 37 °C for 4 h. Then, an aliquot
of the resulting solution was analyzed by ITLC using 1 M HCl/MeOH
(5:95) as eluent, and the distribution of radioactivity on the strips
was monitored using a Berthold LB 505 detector coupled to a radio-
chromatogram scanner. No decomposition was observed since single
peaks were detected at Rf = 0.70–0.85 corresponding to intact Tc1–
Tc4.
Cellular uptake assays of the 99mTc complexes (Tc1–Tc4) were
performed using PC-3, MCF-7 and MCF-7/MDR1cells seeded at a
density of 0.4 million/0.5 mL medium per well in 24-well plates and
allowed to attach overnight. After that period, the medium was
removed and replaced by fresh medium containing approximately
25 kBq/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 that 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
plus the standard deviation of % of total per million of cells.
The cellular uptake of Tc1, Tc3 and Tc4 was also studied on the H69
and H69-LX4 cell lines. For these cells, suspensions were prepared in
the respective culture media at a concentration of 2 × 106 cells/mL.
The suspensions were then incubated for 1 h in humidified atmosphere
with 95% air and 5% CO2 at 37 °C prior to the evaluation of cellular
uptake. The uptake studies were performed after removal of culture
medium followed by incubation with the complexes under study, dilut-
ed in the respective culture media (0.2 MBq/mL) for 15 min, 30 min
and 1 h with constant stirring at 37 ° C (thermostated bath). After the
incubation period, samples of 200 μL were collected (in triplicate) to
microfuge tubes containing 500 μL of cold PBS. The samples were
centrifuged (Eppendorf Centrifuge 5417R) for 3 min at 9400 g. The
supernatant was discarded and the pellet was washed again in 500 μL
of cold PBS. Finally the samples were again centrifuged (10 min,
9400 g) and the activity of the cell pellet was measured in a gamma
counter.
5.8. Alteration of cellular and mitochondrial potential membrane
The assays were performed in PC-3 cells, using buffer solutions
with different concentrations of K+ (5, 60, 120 mM). A normal extra-
cellular concentration (i.e. in the culture medium) of K+ was consid-
ered to be 5 mM [43,44]. For this concentration of K+ (5 mM) a
buffer was used with the following composition (mM): NaCl (145),
KCl (5.4), CaCl2 (1.2), MgSO4 (0.8), NaH2PO4 (0.8), dextrose (5.6),
HEPES (5) and 1% (v/v) fetal bovine serum with a final pH of 7.4.
The solutions of 60 and 120 mM K+ were prepared by replacement
of NaCl by K+-aspartate at the desired concentration, in order to
avoid cytosolic and mitochondrial swelling [34]. For these solutions
the concentration of Ca2+ was further reduced to 0.1 mM and Cl−
was maintained at a concentration of 20 mM. Stock solutions of
valinomycin, nigericin, ouabain and CCCP were prepared in DMSO.
The uptake studies were performed after removal of culture medi-
um followed by incubation of cells with the complex under study. The
99mTc complexes were incubated with buffer solutions of different
concentrations of K+ (5, 60, 120 mM) in a total volume of 500 μL.
When indicated, valinomycin (1 μg/mL) was added to the buffer solu-
tion with 120 mM K+. After 4 h of incubation the procedure followed
was the same as described above for cellular uptake.
In a different set of assays Valinomycin, nigericin, ouabain and
CCCP were added after the incubation of cells with 99mTc complexes
in culture medium for 4 h. After this period, valinomycin (1 μg/mL),
nigericin (5 μg/mL), ouabain (100 μM) and CCCP (5 μM and 10 μM)
were added and the cell further incubated at 37 °C for 15 min. After
this second incubation period, the medium was removed and the pro-
cedure was followed as described for cellular uptake.
5.6. Mitochondrial isolation
Mitochondria were isolated from PC-3 and HEK cells in culture
using a commercial kit (Mitochondria Isolation Kit for Cultured Cells,
Thermo Scientific, Rockford, IL, USA), according to the manufacturer's
instructions. Briefly, after centrifugation (Centrifuge 5804R, Eppendorf)
of a cell suspension with 10–15 × 106 cells for 2 min at 850 g the cell
pellet was resuspended in 450 μL of Reagent A supplemented with a
cocktail of protease inhibitors (Roche, Basel, Switzerland) and trans-
ferred to a microfuge tube. The solution was vortexed at medium
speed for 5 s and was incubated on ice for exactly 2 min. After this
period, 20 μL of Reagent B was added and the sample was vortexed at
maximum speed for 5 s. The sample vial was then incubated on ice
for 5 min, vortexing every minute at maximum speed. Then, 450 μL of
Reagent C supplemented with a cocktail of protease inhibitors was
added and the tube was inverted several times. The sample was
centrifuged at 700 g (Centrifuge 5417R, Eppendorf) for 10 min at 4 °C
and the supernatant was transferred to a new tube and centrifuged
again at 12,000 g for 5 min. The supernatant was discarded (cytosolic
5.9. Evaluation of Pgp expression
Western blot experiments were performed to evaluate the levels
of expression of Pgp in the following cell lines: B16-F1, A375, PC-3,
MCF-7 and MCF-7/MDR1, H69 and H69/Lx4. Cells were lysed in Cell
Lytic-MT Extraction 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 collected for further use. The
total protein content was determined in aliquots of cell lysates by
using the DC Protein Assay Kit (Biorad, Hercules, California, USA),
based on the method of Lowry modified. For the standard calibration
curve ultrapure bovine serum albumin (Biorad) was used. Aliquots of
protein (30 μg) from each sample were analyzed using standard
western blot procedures. Briefly, protein extracts were subjected to
electrophoresis on a 7% SDS-polyacrylamide gel and transferred

C. Moura et al. / Journal of Inorganic Biochemistry 123 (2013) 34–45
45
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Acknowledgments
The authors thank the technical support of Ana Bárbara Pereira.
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/
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