Isostructural Re(i)/99mTc(i) tricarbonyl complexes for cancer theranostics

Article abstract of DOI:10.1039/c5ob00124b

Merging classical organic anticancer drugs with metal-based compounds in one single molecule offers the possibility of exploring new approaches for cancer theranostics, i.e. the combination of diagnostic and therapeutic modalities. For this purpose, we ha

Full text of DOI:10.1039/c5ob00124b

Organic &
Biomolecular Chemistry
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PAPER
99m
Isostructural Re(I)/
Tc(I) tricarbonyl complexes
for cancer theranostics†
Cite this: Org. Biomol. Chem., 2015,
3, 5182
1
a
a
a
a
Patrique Nunes, Goreti Ribeiro Morais, Elisa Palma, Francisco Silva,
a
a
a
a
Maria Cristina Oliveira, Vera F. C. Ferreira, Filipa Mendes, Lurdes Gano,
b,c
b,c,d,e
a
a
Hugo Vicente Miranda, Tiago F. Outeiro,
Isabel Santos and António Paulo*
Merging classical organic anticancer drugs with metal-based compounds in one single molecule offers
the possibility of exploring new approaches for cancer theranostics, i.e. the combination of diagnostic and
therapeutic modalities. For this purpose, we have synthesized and biologically evaluated a series of
9
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Re(I)/
Tc(I) tricarbonyl complexes (Re1–Re4 and Tc1–Tc4, respectively) stabilized by a cysteamine-
based (N,S,O) chelator and containing 2-(4’-aminophenyl)benzothiazole pharmacophores. With the
exception of Re1, all the Re complexes have shown a moderate cytotoxicity in MCF7 and PC3 cancer
cells (IC50 values in the 15.9–32.1 μM range after 72 h of incubation). The cytotoxic activity of the Re com-
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plexes is well correlated with cellular uptake that was quantified using the isostructural
Tc congeners.
There is an augmented cytotoxic effect for Re3 and Re4 (versus Re1 and Re2), and the highest cellular
uptake for Tc3 and Tc4, which display a long ether-containing linker to couple the pharmacophore to the
(N,S,O)-chelator framework. Moreover, fluorescence microscopy clearly confirmed the cytosolic
accumulation of the most cytotoxic compound (Re3). Biodistribution studies of Tc1–Tc4 in mice
confirmed that these moderately lipophilic complexes (log Do/w = 1.95–2.32) have a favorable bioavailabil-
Received 21st January 2015,
Accepted 26th March 2015
ity. Tc3 and Tc4 presented a faster excretion, as they undergo metabolic transformations, in contrast to
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complexes Tc1 and Tc2. In summary, our results show that benzothiazole-containing Re(I)/
Tc(I) tricar-
DOI: 10.1039/c5ob00124b
bonyl complexes stabilized by cysteamine-based (N,S,O)-chelators have potential to be further applied in
the design of new tools for cancer theranostics.
www.rsc.org/obc
detection of β-amyloid (Aβ) aggregates by positron emission
Introduction
tomography (PET) and single-photon emission computed
3
Many benzothiazole derivatives have shown relevant biological tomography (SPECT). The lead compound in these studies
1
1
features for drug development, both for diagnostic and thera- has been the so-called “Pittsburgh compound B” ( C-PIB)
peutic purposes. This class of compounds has proven to be (Fig. 1). Studies in Alzheimer’s disease (AD) patients validated
1
1
particularly useful for the design of radioactive probes for the usefulness of C-PIB as a PET imaging probe for Aβ detec-
nuclear imaging and as cytotoxic drugs for antitumor
1
–3
therapies.
For nuclear imaging, 2-arylbenzothiazole derivatives have a
crucial role in the development of radioprobes for the in vivo
a
Centro de Ciências e Tecnologias e Nucleares, IST, Universidade de Lisboa, Estrada
Nacional 10, 2695-066 Bobadela LRS, Portugal. E-mail: apaulo@ctn.ist.utl.pt
b
Instituto de Medicina Molecular, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal
c
CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa,
Portugal
d
Instituto de Fisiologia, Faculdade de Medicina da Universidade de Lisboa, Lisboa,
Portugal
e
Department of NeuroDegeneration and Restorative Research, University Medical
Center Göttingen, Waldweg 33, 37073 Göttingen, Germany
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
Fig. 1 Chemical structures of clinically relevant benzothiazole
c5ob00124b
derivatives.
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1
1
4,5
tion. However, C-PIB is not approved for clinical use.
ated as SPECT- or MRI-imaging compounds for cancer thera-
1
8
16–18
Nevertheless, structurally related F-labeled congeners, such nostics, respectively.
These first studies led to encouraging
1
8
as F-flutemetamol (Fig. 1), have been recently approved by biological results underlying the relevance of benzothiazole-
the US Food and Drug Administration (FDA) and European containing metal complexes in this field of research. In this
Medicine Agency (EMA) for the visual detection of Aβ burden in context, we have embarked on the synthesis and biological
6
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patients suspected of AD. Several metal-based compounds evaluation of new benzothiazole-containing
Tc(I) and Re(I)
bearing arylbenzothiazole moieties were also evaluated for in vivo tricarbonyl complexes stabilized by a (S,N,O)-donor BFC
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imaging of Aβ plaques. This included the study of
Tc com- derived from cysteamine. Here, we report on the synthesis and
plexes for SPECT imaging and Gd complexes as contrast agents characterization of these new complexes, as well as on their
7–9
for magnetic resonance imaging (MRI). The results for metallic biological evaluation as potential cancer theranostic agents.
complexes were less encouraging if compared with purely organic For the Re complexes, the biological evaluation comprised the
molecules, reflecting their poor ability to penetrate the blood– assessment of their cytotoxic activity against human breast
brain barrier and to reach the β-amyloid plaques.
The 2-arylbenzothiazole scaffold is also promising for the the
development of anticancer drugs. It was shown that such a bromide (MTT) assay, and the visualization of cellular uptake
cancer – MCF7 – and prostate cancer – PC3 – cell lines using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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simple scaffold might afford possible drug candidates upon by fluorescence microscopy. Conversely, the
Tc counter-
the introduction of minor functional group changes in its parts were used for the quantification of the uptake in the
core. Many benzothiazole derivatives have demonstrated same cell lines, and to ascertain the biodistribution and
activity against a wide range of human tumor cell lines, includ- in vivo stability of the complexes in mice.
ing ovarian, breast, prostate lung, renal, and colon cancer cell
1
0–13
lines.
From a large number of tested molecules, the most
explored as an antitumour agent is Phortress (Fig. 1), a 2-(4′-
Results and discussion
Synthesis and characterization of the ligands
aminophenyl)benzothiazole derivative that underwent a Phase
1
0
1
clinical trial.
Merging classical organic anticancer drugs with metal- As mentioned above, we have considered (S,N,O)-donor BFCs
+
based compounds in one single molecule offers the possibility derived from cysteamine to stabilize the fac-[M(CO) ] (M =
3
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of exploring new approaches for cancer theranostics, based on Re,
Tc) core and to be functionalized with the 2-(4′-amino-
the combination of diagnostic and therapeutic modalities. The phenyl)benzothiazole pharmacophore. In the last few years,
metal fragments might confer advantages as theranostic tools, this core has gained an increasing impact in radiopharmaceu-
such as the possibility of exploring a larger diversity of chemi- tical chemistry following the introduction by Alberto and co-
cal structures, which may increase their bioavailability and workers of a convenient and fully aqueous-based kit prepa-
resistance to metabolic transformation. For this purpose, a ration of the organometallic precursors fac-[M(OH ) -
2
3
+
19,20
large variety of d and f transition metals with nuclear or mag- (CO)
3
] .
This so-called tricarbonyl approach has several
netic properties suitable for medical imaging is available, for intrinsic advantages, including the high stability and kinetic
1
4,15
example the aforementioned Tc and Gd.
inertness inherent to Tc(I) and Re(I) tricarbonyl complexes
2
1
In the particular case of technetium, non-radioactive Re when appropriate ligands are used. Previously, we have
complexes are often used as reference compounds to assign shown that the cysteamine-based (S,N,O)-donor ligand 2-(2-
Tc congeners due to the simili- (ethylthio)ethylamino)acetic acid affords low-molecular weight
tude of the chemistry of these two group 7 elements. Hence, and lipophilic
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the chemical identity of the
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Tc(I)/Re(I) complexes with high in vitro and
2
2,23
when designing metal-based cancer theranostic agents, the Re in vivo stability.
We have anticipated that the replacement
complexes can be incorporated into the cytotoxic entity that of the S-terminal ethyl substituent of this chelator by the 2-(4′-
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will exert a therapeutic effect while the
Tc congeners are aminophenyl)benzothiazole pharmacophore would not com-
part of the imaging entity that will enable the in vivo detection promise the coordination capability of the (S,N,O)-donor set
+
99m
of tumor tissues. In addition, there are two beta-emitting towards fac-[M(CO) ] (M = Re,
Tc) and the biological pro-
3
1
86
188
radioisotopes of rhenium ( Re and Re) that present physi- perties of the pharmacophore.
1
4
cal properties suitable for radionuclide therapy. Therefore,
Four new BFCs, L1–L4, were synthesized by linking 2-aryl-
Tc and Re can be seen as a unique match-pair of d-transition benzothiazole pharmacophores to the 3-((2-mercaptoethyl)-
elements that enables the design of radiopharmaceuticals for amino)propanoic acid coordinating unit by using two different
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186/188
diagnostic ( Tc) and therapeutic applications (
Re), aliphatic spacers, displaying or not an ether function
offering further possibilities for cancer theranostics by allow- (Schemes 1 and 2). The synthesis of L1–L4 has been achieved
ing the combination of SPECT imaging with chemo- and/or in a convergent way, using compounds 3 and 4 as common
radiotherapeutic modalities.
intermediates. As depicted in Scheme 1, the 2-aryl benzothia-
Recently, the 2-(4′-aminophenyl)benzothiazole pharmaco- zole rings of 3 and 4 were formed through cyclocondensation
phore was linked to acyclic or macrocyclic bifunctional chela- of the o-aminothiophenols 1 and 2, by reaction with the ade-
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tors (BFCs), which were used to obtain
Tc(I) tricarbonyl quate N-substituted benzaldehyde under basic conditions. For
complexes and Gd(III) complexes. These complexes were evalu- L1 and L2, which do not contain any ether function in their
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Scheme 1 Synthetic route for the preparation of ligands L1 and L2. Conditions: (i) N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde, pyridine,
reflux; (ii) CH Cl , CBr , PPh , RT; (iii) NaOH, EtOH, 2-aminoethanethiol hydrochloride, RT; (iv) CH CN, KI, K CO , ethyl 2-bromoacetate, reflux; (v)
THF, water, NaOH, reflux.
2
2
4
3
3
2
3
Scheme 2 Synthetic route for the preparation of L3 and L4. Conditions: (i) THF, NaH, 1,4-dibromobutane; (ii) NaOH, EtOH, 2-aminoethanethiol
hydrochloride, RT; (iii) CH CN, KI, K CO , ethyl 2-bromoacetate, reflux; (iv) THF, water, NaOH, reflux.
3
2
3
structure, the synthesis involved the brominated derivatives 5 S-alkylation reactions of 2-aminoethanethiol, followed by the
and 6 as intermediates. Compounds 5 and 6 were obtained in N-alkylation of the resulting compounds (7, 8, 13, and 14,
moderate yield (η = 56–64%) from the benzothiazoles 3 and 4 respectively) with ethyl bromoacetate. Finally, basic hydrolysis
using the Appel reaction (Scheme 1). The synthesis of L3 and of the obtained benzothiazole ester derivatives (9, 10, 15, and
L4, having an ether function in the linker between the pharma- 16, respectively) gave the ligands L1–L4. All the compounds
cophore and the S,N,O-coordinating unit, was also achieved were characterized using common spectroscopy techniques
1
13
using brominated intermediates (compounds 11 and 12); 11 ( H and C NMR) and by ESI-MS, which confirmed the pro-
and 12 were obtained in good yield (η = 40–45%) by the posed formulations.
O-alkylation of 3 and 4 with an excess of 1,4-dibromobutane
Synthesis and characterization of the organometallic Re and
(Scheme 2). For all the ligands, the incorporation of the ben-
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Tc complexes
zothiazole moieties into the (S,N,O)-donor coordination unit
was accomplished in a sequential manner, in which the bromi- The synthesis of the Re(I) tricarbonyl complexes Re1–Re4
nated derivatives 5, 6, 11, and 12 acted as electrophiles in was performed by ligand exchange reactions of fac-
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1
Fig. 2 H NMR spectra of complex Re1 at various temperatures in
DMF-d
7
.
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Scheme 3 Synthesis of the Re(I) and
Tc(I) tricarbonyl complexes.
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Conditions: (i) [Re(H
2
O)
3
(CO)
3
]Br, methanol, reflux, 16 h; (ii) [
Tc
(
2 3 3 2
H O) (CO) ], H O, 100 °C, 30 min.
probably contain ligands coordinated in a N,S,O-tridentate
fashion, a coordination mode that was confirmed by X-ray
2
3
diffraction analysis in the case of Re(CO)
3
(NSO).
In aqueous solution and at 100 °C, L1–L4 readily reacted
[
(
Re(H O) (CO) )]Br with L1–L4 in refluxing methanol
2 3 3
Scheme 3). After adequate purification, complexes Re1–Re4
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+
with fac-[ Tc(CO)
3
(OH
2
)
3
]
affording complexes Tc1–Tc4,
respectively (Scheme 3). The reactions are almost quantitative
were recovered as yellow microcrystalline solids in moderate to
high yield (57–81%). Their characterization was performed
using common spectroscopy techniques (IR, H and
NMR), ESI-MS, HPLC and elemental analysis.
−4
(
>95%) for final concentrations of L1–L4 as low as 10 M,
1
13
complexes Tc1–Tc4 being the unique species formed. Their
chemical identity was confirmed by comparison of their HPLC
profiles with those of the corresponding rhenium complexes,
as exemplified for Tc1/Re1 in Fig. 3.
The characterization of Tc1–Tc4 comprised also the evalu-
ation of their lipophilicity, which was assessed by the measure-
ment of the respective log Do/w values (n-octanol/0.1 M,
phosphate buffered saline (PBS), pH 7.4) using the “shake-
flask” method. The log Do/w values and the HPLC retention
times of Tc1–Tc4 are shown in Table 1. All the complexes are
moderately lipophilic with log Do/w values spanning in a rather
narrow range, from 1.98 to 2.32, suggesting that the presence
of the ether linkage balances the increase of lipophilicity
C
The positive ESI mass spectra obtained for Re1–Re4 showed
the presence of prominent peaks corresponding to the respect-
ive [M + H] molecular ions; unlike Re1 and Re2, intense peaks
that were assigned to [M + Na] were also observed in the mass
spectra of Re3 and Re4. This difference certainly reflects the
presence of an ether function in the aliphatic spacers of L3
and L4, which might enhance the interaction with the sodium
cation. The IR spectroscopic data obtained for all the com-
plexes corroborated the presence of the fac-[Re(CO)
+
+
+
3
]
core,
detecting two strong bands assignable to ν(CvO) in the
1
893–2028 cm− range. Moreover, the HPLC analysis of Re1–
1
Re4 showed that the complexes were of high purity, as only
single peaks were detected in the respective chromatograms.
The chemical characterization of Re1–Re4 by NMR spec-
1
troscopy was more demanding. At room temperature, the H
NMR spectra of these complexes in DMF-d displayed a set of
7
relatively broad and ill-defined resonances in the area of the
methylenic and methyl protons, ranging from 1.71 to
3
.96 ppm. The aromatic protons from the 2-(4′-aminophenyl)-
benzothiazole pharmacophore gave rise to better defined and
well resolved signals that appear between 6.90 and 8.10 ppm.
The broadness of the resonances from the aliphatic protons
suggested that complexes Re1–Re4 undergo dynamic processes
1
in solution, as confirmed for Re1 by variable temperature H
NMR studies (Fig. 2). The dynamic behavior observed for Re1–
Re4 is most probably due to the occurrence of pyramidal inver-
sion at the coordinated sulfur atom, as we have previously
observed for the congener Re(I) tricarbonyl complex with 2-(2-
(
(
ethylthio)ethylamino)acetic acid (herein designated as Re-
CO) (NSO)). These findings indicate that Re1–Re4 most (radiometric detection).
3
Fig. 3 HPLC chromatograms of complexes Re1 (UV detection) and Tc1
2
2
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Table 1 HPLC retention times and log Do/w of complexes Tc1–Tc4
a,b
Re3, was assessed by fluorescence microscopy in MCF7 and
PC3 human cancer cells. For comparative purposes, the
studies were also performed for the respective ligand L3. Com-
pounds L3 and Re3 were detected by the emission of blue fluo-
Complex
r
t
(min)
log Do/w
Tc1
Tc2
Tc3
Tc4
24.4 (23.8)
24.9 (24.1)
24.0 (23.3)
26.0 (25.9)
1.98 ± 0.02
2.01 ± 0.05 rescence and the nucleus stained with propidium iodide (red
1.95 ± 0.01
emission).
2.32 ± 0.04
As shown in Fig. 4, both complexes Re3 and L3 (to a lesser
a
3
Using a gradient of aqueous 0.1% CF COOH and methanol as the
extent) were detected in the cytoplasm of both cell lines. No
detectable fluorescence in the blue channel was observed for
the DMSO control, confirming that the detected blue emission
is from the compounds L3 and Re3, and not due to the un-
specific signal.
b
solvent. The values in parentheses are the retention times for the Re
congeners.
caused by the larger number of methylenic groups in the
linker.
The cell entrance/uptake seems to be reduced for L3 when
compared with Re3, as less fluorescence was detected from
cells incubated with L3 if compared with those incubated with
Re3. It is important to mention that in Fig. 4 all the images
were collected using the same conditions, in particular the
acquisition time. A second set of images was collected with a
longer acquisition time to further evaluate the cellular uptake
and localization of L3 (Fig. 5), showing intracellular cyto-
plasmic accumulation.
In addition, the fluorescence spectra obtained for L3 and
Re3 (at the same concentration) have shown no detectable
differences in the maximum wavelength of excitation and
emission (Fig. 5). Moreover, these experiments have shown
that L3 is a slightly more efficient fluorophore than Re3.
Hence, the more intense fluorescence detected in the cells
incubated with Re3 is not due to its intrinsic fluorescence
Biological evaluation of the Re complexes: cytotoxicity assays
and microscopy studies
The cytotoxic activity of the ligands and respective Re com-
plexes was tested on human breast cancer – MCF7 – and pros-
tate cancer – PC3 – cell lines. These cancer cells were treated
with decreasing concentrations (200–0.002 μM) of the different
compounds and incubated for 72 h at 37 °C. All the tested
compounds were first solubilized in DMSO and diluted in the
cell media. The percentage of DMSO never exceeded 1%, a
non-toxic concentration. After the treatment, the cellular viabi-
lity was assessed by the MTT assay. The inhibition of growth
(%) was calculated by correlation with vehicle-treated cells.
The IC50 values, expressed in μM concentrations, are average ±
standard deviations of 3 independent experiments with 4 repli-
cates each, and the results are presented in Table 2.
In general, the complexes Re2–Re4 exhibited higher
cytotoxicity than the respective ligands L2–L4 in the MCF7
breast and PC3 prostate cell lines. In contrast, in these cell
lines L1 is more cytotoxic than Re1. From all the studied com-
pounds, the complexes Re3 and Re4 were shown to be the
most potent, with IC50 values between 15.9 ± 1.1 and 32.1 ±
1
.2 μM in both cell lines.
Taking advantage of the fluorescence emission character-
istics of the benzothiazole unit of the newly synthesized com-
pounds, the cellular uptake of the most cytotoxic Re complex,
Table 2 Cytotoxicity of L1–L4 and Re1–Re4 in MCF7 breast and PC3
prostate cancer cells
a
IC50 (µM)
Compound
MCF7
PC3
L1
Re1
L2
Re2
L3
Re3
L4
25.6 ± 1.4
90 ± 1.2
32.5 ± 1.1
94 ± 1.3
89 ± 1.1
30.6 ± 1.0
95 ± 1.2
28.8 ± 1.1
71.3 ± 1.1
32.1 ± 1.2
241 ± 1.8
30.7 ± 1.0
52.2 ± 1.1
15.9 ± 1.1
39.3 ± 1.1
16.3 ± 1.2
Fig. 4 Fluorescence microscopy evaluation of the uptake of L3 and
Re3 (blue) in human MCF7 and PC3 cells. Nuclei (red) were stained with
propidium iodide. Scale bar, 10 μm.
Re4
a
IC50: the concentration that causes 50% reduction of the cell growth.
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properties. Altogether, these data indicate that Re3 is able to
enter into cancer cell lines more easily than the corresponding
ligand L3. Eventually, this might explain the highest cyto-
toxicity that has been found for Re3 in the MCF7 and PC3
cancer cell lines, in comparison with the respective ligand.
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Biological evaluation of
biodistribution studies
Tc complexes: cellular uptake and
The microscopy studies performed for Re3 have confirmed
that this complex can reach the cytoplasm of MCF7 breast and
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PC3 prostate human cancer cells. The isostructural
Tc
complex, Tc3, was used to quantify the cellular uptake in the
same cell lines. These studies were extended to the other radio-
active complexes (Tc1, Tc2 and Tc4) aiming to correlate their
cellular uptake with the cytotoxicity exhibited by the corres-
ponding Re complexes. The breast and prostate cancer cell
lines were treated with Tc1–Tc4 for different times (0 to 18 h)
and the results show that complexes Tc1–Tc4 display a high
cellular uptake in both cell lines, in a time-dependent manner,
with values at 18 h in the range of 14–29% and 23–48% for
MCF7 and PC3 cells, respectively (Fig. 6). The highest uptake
was observed in the PC3 cells, in particular for the complexes
Tc3 and Tc4 with values of 38.6% and 31.5% at 5 h and values
of 47.1% and 47.9% at 18 h, respectively. These results demon-
strate that the complexes Tc3 and Tc4 display the highest cel-
lular uptake in the MCF7 and PC3 cells, accumulating in both
cell lines over time. This trend seems to be in agreement with
the highest cytotoxicity of the Re congeners (Re3 and Re4), par-
ticularly in the MCF7 breast cancer cell line.
As discussed before, all complexes are lipophilic with
log Do/w values within the relatively narrow range of 1.98–2.32.
Therefore, the lipophilicity is not expected to be the major
cause of the large differences observed between the cellular
uptake of Tc1–Tc4. This is clearly corroborated by the fact that
Tc1 (log P = 1.98 ± 0.02) and Tc3 (log P = 1.95 ± 0.02), having
almost equal log P values, present a large difference in the cel-
lular uptake values, which for Tc3 is as high as 30.9% of the
Fig. 5 Emission and excitation spectra of L3 (a) and Re3 (b). Fluo-
rescence microscopy evaluation of human MCF7 and PC3 cancer cell
uptake of L3 (blue) (c). Nuclei (red) were stained with propidium iodide.
Note: images were collected with higher acquisition times than those applied activity/million cells in the PC-3 cell line versus 13.3%
presented in Fig. 4. Scale bar, 10 μm.
for Tc1 at 5 h.
Fig. 6 Cellular uptake of Tc1–Tc4 in human MCF7 breast and PC3 prostate cells, expressed as a percentage of total radioactivity per million cells.
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Table 3 Biodistribution data (%ID g− ) of Tc1–Tc4 at 2 and 60 min post-injection (p.i.) in CD-1 mice
1
ID g− ± SD
1
%
Tc1
Tc2
Tc3
Tc4
Organ
2 min
60 min
2 min
60 min
2 min
60 min
2 min
60 min
Blood
Liver
6.40 ± 1.10
43.3 ± 1.90
3.10 ± 0.40
3.90 ± 0.20
5.40 ± 0.60
7.00 ± 1.00
21.6 ± 2.90
1.20 ± 0.20
1.50 ± 0.20
1.20 ± 0.40
0.30 ± 0.08
0.58 ± 0.11
37.5 ± 1.60
19.5 ± 1.70
0.50 ± 0.20
0.90 ± 0.30
1.10 ± 0.20
2.70 ± 0.20
0.60 ± 0.10
0.46 ± 0.09
2.60 ± 0.70
0.03 ± 0.01
7.00 ± 0.50
19.9 ± 2.60
1.40 ± 0.40
3.70 ± 0.03
3.80 ± 0.70
13.9 ± 0.70
11.8 ± 4.20
0.90 ± 0.30
1.30 ± 0.30
0.69 ± 0.10
0.19 ± 0.04
1.00 ± 0.09
15.2 ± 2.40
11.5 ± 3.20
0.78 ± 0.01
0.90 ± 0.10
3.00 ± 0.80
1.90 ± 0.80
0.55 ± 0.08
0.45 ± 0.06
1.30 ± 0.30
0.04 ± 0.00
8.00 ± 2.40
35.8 ± 1.60
3.90 ± 0.50
2.90 ± 0.60
3.70 ± 0.80
3.50 ± 1.00
14.9 ± 2.20
0.80 ± 0.30
1.40 ± 0.20
0.90 ± 0.60
0.20 ± 0.04
0.70 ± 0.20
11.3 ± 1.20
10.3 ± 1.20
0.26 ± 0.03
0.42 ± 0.08
0.70 ± 0.20
2.00 ± 0.50
0.29 ± 0.07
0.19 ± 0.03
1.50 ± 0.50
0.02 ± 0.01
5.20 ± 0.90
35.8 ± 4.40
2.40 ± 0.30
5.20 ± 0.90
4.40 ± 0.20
9.00 ± 1.00
13.2 ± 0.70
1.40 ± 0.10
1.96 ± 0.07
1.80 ± 0.50
0.40 ± 0.10
0.84 ± 0.05
29.5 ± 0.60
17.2 ± 0.50
0.70 ± 0.10
1.10 ± 0.20
3.70 ± 0.60
2.10 ± 0.40
0.67 ± 0.06
0.54 ± 0.04
3.60 ± 0.70
0.05 ± 0.01
Intestine
Spleen
Heart
Lungs
Kidney
Muscle
Skeletal
Stomach
Brain
Excretion (% IA)
2.70 ± 0.50
3.60 ± 2.40
18.4 ± 1.70
12.4 ± 3.40
The most striking structural differences in complexes Tc1– I.A. At 1 h p.i., most of the activity is retained in the liver and
Tc4 are related to the linkers used to attach the 2-arylben- intestine due to the predominantly hepatobiliary excretion of
zothiazole pharmacophore to the 3-((2-mercaptoethyl)amino)- these moderately lipophilic compounds. Despite their neu-
propanoic acid coordinating unit. A shorter ethylenic linker trality and lipophilicity, Tc1–Tc4 showed a negligible brain
has been used in complexes Tc1 and Tc2, which have shown a uptake (<0.40% ID g− ) confirming their inability to cross the
much lower cellular uptake than Tc3 and Tc4, containing a blood–brain barrier in mice.
1
longer 2-n-butoxyethyl linker. The use of a longer linker is
The in vivo stability of Tc1–Tc4 has been assessed by HPLC
expected to minimize the interference of the organometallic analysis of the urine and plasma of mice injected with these
fragment in molecular interactions involving the pharmaco- complexes. At 2 min p.i., most of the radioactivity detected in
phore and biologically relevant targets. It is worthwhile men- the plasma corresponds to the intact complexes (Fig. 7).
tioning that many benzothiazole derivatives like the parent However, the complexes with an ether-containing longer
compound 2-(4′-aminophenyl)-benzothiazole and its deriva- linker, Tc3 and Tc4, have a higher tendency to undergo meta-
tives bearing 3′ substituents undergo a selective uptake and bolic transformations, as indicated by the HPLC chromato-
biotransformation only in sensitive cancer cell lines, such as grams obtained for the plasma at 1 h p.i. As exemplified for
MCF-7 breast cancer cells. In these sensitive cell lines, the Tc4 in Fig. 7, a significant part of the circulating activity still
compounds bind to the aryl hydrocarbon receptor (AhR) with corresponds to the intact complex but two metabolites were
AhR translocation to the nucleus and subsequent induction of detected at retention times of 17.2 and 18.7 min, respectively.
the expression and activation of the cytochrome P450 isoform These metabolites are also the major radiochemical species
CYP1A1. In contrast, insensitive cells like the prostate carci- that are present in the HPLC chromatograms of the urine of
noma PC3 cells do not show AhR translocation and CYP1A1 mice injected with Tc4, at 1 h p.i. (data not shown). We did
activation upon treatment with the same benzothiazole deriva- not identify these metabolites but their formation might be
12,13,24
tives.
We did not fully investigate the mechanisms related to transformations involving the ether function from
involved in the cellular uptake of Tc1–Tc4 and in the cytotoxic the aliphatic linker used to couple the benzothiazole pharma-
activity of the congener Re1–Re4, but the higher uptake cophore with the (N,S,O) chelator framework in complexes Tc3
9
9m
observed for the
Tc complexes in the PC3 cell line in and Tc4. For instance, the ether function can be involved in
comparison with the MCF7 cell line and similar cytotoxic O-dealkylation processes, followed by oxidation of the resulting
9
9m
activity of the Re complexes in both cell lines indicate that the alcohol functions, as we have recently shown for
interaction with AhR is most likely not responsible for the bio- bonyl complexes with ether-containing pyrazolyl derivatives.
logical activity exhibited. The formation of these metabolites in vivo can certainly justify
Tc(I) tricar-
2
5
Finally, biodistribution studies of complexes Tc1–Tc4 were that complexes Tc3 and Tc4 display a faster excretion rate than
performed in CD-1 mice to assess their pharmacokinetic Tc1 and Tc2.
profile and in vivo stability. The biodistribution data obtained
for Tc1–Tc4 at 2 min and 1 h post intravenous injection (p.i.),
expressed in percent of the injected dose per gram of tissue
(
Conclusions
%ID g− ), are presented in Table 3.
1
All complexes show a relatively fast blood clearance but a The functionalization of the 3-((2-mercaptoethyl)amino)propa-
rather low rate of excretion that varied between 2.70 and 18.4% noic acid coordinating unit with the 2-(4′-aminophenyl)benzo-
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Experimental section
General information
All chemicals and solvents were of reagent grade and were
used without purification unless stated otherwise. 2-Amino-5-
methylbenzenethiol (2) was prepared as described in the litera-
2
6
ture. fac-[Re(H
2
O)
3
(CO)
3
]Br was synthesized according to the
Na[ TcO4] was eluted from a commercial
Mo/ Tc generator, using a 0.9% saline solution. The fac-
2
7
99m
literature.
9
9
99m
9
9m
+
[
Tc(H
2 3 3
O) (CO) ] precursor was obtained by labelling of a
9
9m
Isolink®-kit with Na[ TcO4], following the procedure
described elsewhere. The NMR spectra were recorded on a
Varian Unity 300 NMR spectrometer at frequencies of 300 MHz
2
0
Fig. 7 In vivo stability studies of complexes Tc2 and Tc4. HPLC chro-
matograms of: (a) injected preparation; (b) plasma collected at 2 min p.i.;
1
13
(
H) and 75 MHz ( C). Chemical shifts are reported in parts
1
13
per million. H and C NMR chemical shifts were referenced
with the residual solvent resonances relative to tetramethyl-
silane. FTIR spectra were recorded using KBr pellets on a
Bruker, Tensor 27 spectrometer. Electrospray ionisation mass
(
c) plasma collected at 1 h p.i.
thiazole pharmacophore, using two different aliphatic spacers spectrometry (ESI-MS) was performed on a QITMS instrument
containing or not an ether function, led to a new family of in positive and negative ionization mode. Thin-layer chromato-
benzothiazole-containing chelators: L1–L4. These chelators graphy (TLC) was performed on precoated silica plates 60 F254
were successfully applied in the synthesis of M(I) (M = Re, (Merck). Visualization of the plates was carried out using UV
9
9m
Tc) tricarbonyl complexes (Re1–Re4 and Tc1–Tc4), which light (254 and 365 nm) and/or an iodine chamber. Gravity
were biologically evaluated as isostructural tools for cancer column chromatography was carried out on silica gel (Merck,
9
9m
theranostics.
70–230 mesh). HPLC analysis of the Re and
Tc complexes
The Re complexes have shown a moderate cytotoxic activity was performed on a PerkinElmer LC pump coupled to an LC
against PC3 and MCF7 human tumor cells (IC50 values <50 μM tuneable UV/Vis detector and to a Berthold LB-507A radio-
after 72 h of incubation), with the exception of Re1 that metric detector, using an analytical C18 reversed-phase
showed relatively high IC50 values (>50 μM). In general, the Re column (Nucleosil 100-10, 250 × 3 mm) with a flow rate of 1 ml
−
1
complexes are more cytotoxic than the corresponding ligands, min . UV detection, 254 nm. Eluents: A – aqueous 0.1%
which is most probably due to the better ability of the com- CF COOH solution, B – MeOH. The HPLC analysis was per-
3
plexes to enter into the cells, as confirmed by fluorescence formed with gradient elution, using the following method:
microscopy studies in the case of Re3/L3.
0–8 min, 100% A; 8–8.1 min, 100%–75% A; 8.1–14 min, 75% A;
There is a clear influence of the linker on the cytotoxicity of 14–14.1 min, 75%–66% A; 14.1–25 min, 66%–0% A; 25–30 min,
the complexes, those which display the ether-containing 0% A; 30–30.1 min, 0–100% A; 30.1–35 min, 100% A.
linker, i.e. Re3 and Re4, being the most active. The cytotoxicity
Synthesis of the ligands
activity of the Re complexes is well correlated with the cellular
9
9m
uptake, which has been quantified using the
Tc1–Tc4. Interestingly, the nature of the linker also strongly zole (3). A solution of o-aminothiophenol (557 mg, 4.5 mmol)
influences the in vivo behavior of the complexes, as Tc3 and and
N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde
Tc4 are more prone to metabolic transformations and display (660 mg, 3.7 mmol) in pyridine (6 mL) was refluxed for 3 h.
a faster excretion rate in mice.
After cooling at room temperature the intense yellow suspen-
Tc congeners,
2-[N-Methyl-N-(2′-hydroxyethyl)-4′-aminophenyl]-benzothia-
In summary, the moderate cytotoxicity exhibited by the Re sion was neutralized with a 3 M HCl solution and the solvent
complexes, together with the remarkably high cellular uptake was concentrated. The reaction crude was taken in water
9
9m
and the reasonably good bioavailability presented by the
Tc (50 mL) and extracted with CH Cl (3 × 30 ml). The organic
2 2
congeners, show that the benzothiazole-containing Re(I)/ phase was dried over Na SO , filtered and evaporated. The
2
4
9
9m
Tc(I) tricarbonyl complexes described here hold promise to residue was subjected to column chromatography on silica-gel
be further applied in the design of new tools for cancer thera- (CH Cl –MeOH, 98 : 2) to give 3 (510 mg, 49%); R = 0.25
nostics. The marked influence of the linker on the biological (n-hexane–AcOEt 1 : 1); H NMR (CDCl , 300 MHz): δ 1.78 (bs,
2
2
f
1
3
fate of the complexes and its easy structural modification, as OH, 1H), 3.07 (s, 3H), 3.58 (t, 2H, J = 5.7 Hz), 3.86 (t, 2H, J =
3
3
3
well as the different possibilities available to attach the 2-(4′- 5.7 Hz), 6.79 (d, 2H, J = 8.7 Hz), 7.29 (dd, 1H, J = 8.1 Hz, J =
3
3
3
aminophenyl)benzothiazole pharmacophore (e.g. N-alkylation 8.1 Hz), 7.42 (dd, 1H, J = 8.1 Hz, J = 8.1 Hz), 7.82 (d, 2H, J =
3
3
13
vs. amide-forming reactions) to the cysteamine based (N,S,O)- 8.1 Hz), 7.94 (d, 2H, J = 8.7 Hz), 7.98 (d, 2H, J = 8.1 Hz);
C
chelator are expected to help in the search for better perform- NMR (CDCl , 75 MHz): δ 39.05, 54.67, 60.17, 111.98 (2C),
3
ing compounds and on the elucidation of structure–activity 121.37 (2C), 122.15, 124.38, 126.14, 129.07 (2C); ESI/MS
+
relationships (SAR).
C H N OS (284) m/z 284.9 [M + H] (100%).
1
6
16 2
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3
13
6
-Methyl-2-[N-methyl-N-(2′-hydroxyethyl)-4′-aminophenyl]-
J = 7.8 Hz); C NMR (CDCl
3
, 75 MHz): δ 28.5, 36.4, 38.6, 41.1,
benzothiazole (4). Compound 4 was synthesized and purified 52.4, 111.4 (2C), 121.3, 121.5, 122.2, 124.2, 125.9, 128.9 (2C),
as described above for compound 3, starting from 4-methyl-o- 134.4, 150.3, 154.2, 168.5.
aminothiophenol (320 mg, 2.30 mmol). Yield: 60% (329 mg,
6-Methyl-2-[N-methyl-N-(2′-(2″-aminoethylthio)ethyl)-4′-ami-
1
1
.38 mmol); R = 0.43 (petroleum ether–AcOEt 1 : 1); H NMR nophenyl]-benzothiazole (8). Compound 8 was synthesized
f
(
CDCl
3
, 300 MHz): δ 2.45 (s, 3H), 3.06 (s, 3H), 3.52 (t, 2H, J = and purified as described above for 7, starting from compound
3
5
1
2
6
.7 Hz), 3.85 (t, 2H, J = 5.7 Hz), 6.78 (d, 2H, J = 9 Hz), 7.23 (d, 6 (200 mg, 0.55 mmol). Yield: 35% (68 mg, 0.19 mmol); R
f
=
3
3
1
H, J = 8.1 Hz), 7.61 (s, 1H), 7.85 (d, 1H, J = 8.1 Hz), 7.91 (d, 0.48 (CHCl –MeOH 4 : 1); H NMR (CDCl , 300 MHz): δ 2.42 (s,
3
3
3
13
H, J = 9 Hz); C NMR (CDCl
0.1, 111.9 (2C); 121.2, 121.6, 127.7, 128.9, 134.5, 151.5, 168.
-[N-Methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-benzothiazole
3
, 75 MHz): δ 21.5, 39.0, 54.7, 3H), 2.65 (t, 2H, J = 7.5 Hz), 2.88 (m, 2H), 2.93 (s, 3H), 3.14 (m,
3
2H), 3.50 (t, 2H, J = 7.5 Hz), 6.62 (d, 2H, J = 9 Hz), 7.19 (d, 1H,
3
13
2
J = 8.4 Hz), 7.55 (s, 1H), 7.82 (m, 3H); C NMR (CDCl3,
(5). To a solution of 3 (0.51 g, 1.8 mmol) in dichloromethane 75 MHz): δ 21.4, 21.3, 28.5, 29.5, 38.5, 39.2, 52.1, 111.7 (2C),
(10 mL) was added carbon tetrabromide (0.65 g, 1.9 mmol) at 121.2, 121.6, 127.6, 128.9 (2C), 134.4, 150.3, 152.1, 167.8,
0
°C followed by triphenylphosphine (0.517 g, 1.8 mmol). The 176.7.
reaction mixture was stirred for 3 h and, thereafter, the solvent Ethyl
2-[2′-(2″-((4″-(benzo[d]thiazol-2-yl)phenyl)(methyl)-
was concentrated. The residue was subjected to column amino)ethylthio)ethylamino] acetate (9). To a solution of 7
chromatography on silica gel (EtOAc–n-hexane 1 : 6) to afford (100 mg, 0.29 mmol), KI (1.2 mg, 7.3 μmol), and K CO
2
3
5
. Yield: 56% (350 mg, 1.0 mmol) as an yellow oil; R
f
= 0.46 (20 mg, 0.15 mmol) in anhydrous CH
, 300 MHz): δ 3.09 (s, ethyl 2-bromoacetate (8.2 μL, 0.145 mmol) under a nitrogen
H), 3.48 (t, 2H, J = 7.5 Hz), 3.80 (t, 2H, J = 7.5 Hz), 6.73 (d, 2H, atmosphere, and the reaction mixture was refluxed overnight.
3
CN (10 mL) was added
1
(AcOEt–n-hexane 1 : 2); H NMR (CDCl
3
3
3
3
3
J = 8.7 Hz), 7.30 (dd, 1H, J = 8.1 Hz, J = 8.1 Hz), 7.43 (dd, 1H, Hence the solvent was concentrated. The mixture was taken in
J = 8.1 Hz, J = 8.1 Hz), 7.83 (d, 1H, J = 8.1 Hz), 7.96 (d, 2H, water (30 ml) and extracted with CH
J = 8.7 Hz), 8.01 (d, 1H, J = 8.1 Hz); C NMR (CDCl3, organic phase was dried with Na SO , filtered, and the filtrate
3
3
3
3
2
Cl
2
(3 × 30 ml). The
3
13
2
4
7
1
5 MHz): δ 27.89, 38.87, 54.07, 111.56 (2C), 121.38, 122.27, was evaporated. The residue was subjected to column chrom-
24.41, 126.12, 129.17 (2C), 150.11; ESI/MS C16 15BrN S (346) atography on silica gel (CHCl –MeOH 95 : 5) to provide 9.
H
2
3
+
+
+
m/z 347.0 [M + H] (97%), 347.9 [M + H] (20%), 348.9 [M + H]
Yield: 58%. (73 mg, 0.17 mmol) as a light yellow oil; R = 0.55
f
+
1
(100%), 349.9 [M + H] (20%).
(CHCl
3
–MeOH 93 : 7); H NMR (CDCl
3
, 300 MHz): δ 1.19 (t,
6
-Methyl-2-[N-methyl-N-(2′-fluoroethyl)-4′-aminophenyl]-ben- 3H, J = 7.2 Hz), 2.68 (m, 4H), 2.77 (t, 2H, J = 6.3 Hz), 3.00 (s,
zothiazole (6). Compound 6 was synthesized and purified as 3H), 3.36 (s, 2H), 3.55 (t, 2H, J = 6.2 Hz), 4.12 (q, 2H, J = 7.2
3
4
3
described above for 5, starting from compound 4 (300 mg, Hz), 6.67 (d, 2H, J = 9.0 Hz), 7.23 (ddd, 1H, J = 1.2 Hz, J =
3
2
3
3
1
(
3
f
.00 mmol). Yield: 64% (231 mg, 0.64 mmol); R = 0.39 8.1 Hz, J = 7.5 Hz), 7.36 (ddd, 1H, J = 1.2 Hz, J = 8.1 Hz, J =
n-hexane–AcOEt 1 : 4); H NMR (CDCl , 300 MHz): δ 2.46 (s, 8.1 Hz), 7.77 (d, 1H, J = 8.1 Hz), 7.88 (d, 2H, J = 9.0 Hz), 7.90
3
H), 3.09 (s, 3H), 3.48 (t, 2H, J = 7.5 Hz), 3.80 (t, 2H, J = 7.5 (d, 1H, J = 7.5 Hz); C NMR (CDCl , 75 MHz): δ 14.0, 28.3,
3
Hz), 6.73 (d, 2H, J = 8.7 Hz), 7.24 (d, 1H, J = 8.2 Hz), 7.62 (s, 32.4, 38.4, 47.9, 50.3, 52.2, 60.6, 111.3 (2C), 121.1, 121.3, 122.0,
1
3
3
3
13
3
3
3
3
13
1
H), 7.87 (d, 1H, J = 8.2 Hz), 7.95 (d, 2H, J = 8.7 Hz);
NMR (CDCl , 75 MHz): δ 21.1, 27.7, 38.2, 53.4 (CH ), 111.0 (CvO).
2C), 120.8, 121.4, 121.7, 127.1, 128.4 (2C), 133.9, 134.2, 149.4, Ethyl 2-[2′-(2″-((4′′′-(6″″-methylbenzo[d]thiazol-2-yl)phenyl)-
(methyl)amino)ethylthio)ethylamino] acetate (10). Compound
-[N-Methyl-N-(2′-(2″-aminoethylthio)ethyl)-4′-aminophenyl]- 10 was synthesized and purified as described above for 9, start-
C
124.0, 125.7, 128.8 (2C), 134.2, 150.2, 154.1, 168.4, 171.9
3
2
(
1
+
51.9, 166.9; ESI/MS C H BrN S (362) m/z 363.2.0 [M + H] .
17 17 2
2
benzothiazole (7). To a solution of 2-aminoethanethiol hydro- ing from compound 8 (55 mg, 0.15 mmol). Yield: 55% (37 mg,
1
chloride (296 mg, 2.55 mmol) in anhydrous EtOH (8 mL) was 0.08 mmol); R = 0.48 (CHCl –MeOH, 97 : 3); H NMR (CDCl ,
f
3
3
added NaOH (163 g, 4 mmol) under a nitrogen atmosphere. 300 MHz): δ 1.24 (t, 3H, J = 6.9 Hz), 2.44 (s, 3H), 2.72 (m, 4H),
After 1 h, compound 5 (448 mg, 1.29 mmol) was added and 2.82 (t, 2H, J = 5.7 Hz), 3.03 (s, 3H), 3.40 (s, 2H), 3.59 (t, 2H, J =
3
the reaction mixture was stirred for additional 20 h at room 7.2 Hz), 4.16 (q, 2H, J = 6.9 Hz), 6.70 (d, 2H, J = 9.0 Hz), 7.23
3
3
temperature. Then, the solvent was concentrated. The pallid (d, 1H, J = 8.4 Hz), 7.60 (s, 1H), 7.83 (d, 1H, J = 8.4 Hz), 7.91
3
13
brown residue was taken in water (25 mL) and the pH was (d, 2H, J = 9.0 Hz); C NMR (CDCl
adjusted to 7 with a 1 M HCl solution. The aqueous phase was 28.6, 32.6, 38.6, 48.2, 50.3, 52.5, 60.8, 111.5 (2C), 121.1, 121.8,
extracted with CH Cl (4 × 25 mL), dried over Na SO , filtered 127.4, 128.8 (2C), 134.2, 134.6, 150.2, 152.4, 167.5, 172.1
and the filtrate was concentrated. The residue was subjected to (CvO).
3
, 75 MHz): δ 14.2, 21.4,
2
2
2
4
column chromatography on silica gel (CHCl –MeOH 70 : 30) to
2-[N-Methyl-N-(2′-(4′-bromobutoxy)ethyl)-4′-aminophenyl]-
3
afford the desired amine as an oil. Yield: 76% (335 mg, benzothiazole (11). To a suspension of NaH (525 mg,
1
0
3
3
=
7
.98 mmol); R
f
= 0.64 (CHCl
3
–MeOH 3 : 1); H NMR (CDCl
3
,
13.2 mmol) in anhydrous THF (40 mL) was added 3 (2.0 g,
00 MHz): δ 2.68 (m, 4H), 2.87 (t, 2H, J = 6.9 Hz), 3.04 (s, 3H), 8.05 mmol) under a nitrogen atmosphere. After 30 min, 1,4-
.59 (t, 2H, J = 6.9 Hz), 6.71 (d, 2H, J = 8.7 Hz), 7.28 (dd, 1H, J dibromobutane (5.6 mL, 47.3 mmol) was added and the solu-
7.8 Hz, J = 7.2 Hz), 7.41 (dd, 1H, J = 7.2 Hz, J = 7.8 Hz), tion was left on vigorous stirring for 3 days at room tempera-
.82 (dd, 1H, J = 7.8 Hz), 7.93 (d, 2H, J = 8.7 Hz), 7.95 (d, 1H, ture. Thereafter, the solvent was concentrated and the reaction
3
3
3
3
3
3
3
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4
3
3
residue was subjected to column chromatography on silica gel 8.1 Hz), 7.80 (dd, 1H, J = 0.6 Hz, J = 7.8 Hz), 7.90 (d, 2H, J =
3
13
(
n-hexane–AcOEt 1 : 1) to afford 11. Yield: 45% (1.518 g, 9.0 Hz), 7.94 (d, 1H, J = 8.1 Hz); C NMR (CDCl , 75 MHz):
3
1
3
3
4
.62 mmol); R
f
= 0.54 (CHCl
3
–MeOH 99 : 1); H NMR (CDCl
3
,
δ 13.9, 25.9, 28.5, 31.2, 31.4, 38.8, 47.4, 49.8, 51.8, 60.6, 67.8,
00 MHz): δ 1.67 (m, 2H), 1.89 (m, 2H), 3.04 (s, 3H), 3.41 (m, 70.5, 111.2 (2C), 120.8, 121.1, 121.9, 123.9, 125.7, 128.6 (2C),
H), 3.58 (s, 4H), 6.68 (d, 2H, J = 9.0 Hz), 7.23 (dd, 1H, J = 7.2 134.1, 150.8, 154.0, 168.5, 171.5 (CvO); ESI/MS C H N O S
35 3 3 2
Hz, J = 7.8 Hz), 7.37 (d, 1H, J = 7.2 Hz, J = 8.1 Hz), 7.77 (d, (501.2) m/z 502.4 [M + H] .
Ethyl 2-[4′-((6″-methyl)benzo[d]thiazol-2-yl)phenyl]-5-oxa-10-
Hz); C NMR (CDCl , 75 MHz): δ 28.2, 29.6, 33.7, 39.2, 52.2, thia-2,13-diazapentadecan-15-oate (16). Compound 16 was
3
3
2
6
3
3
3
+
3
3
3
1
H, J = 7.8 Hz), 7.88 (d, 2H, J = 9.0 Hz), 7.92 (d, 1H, J = 8.1
1
3
3
6
8.2, 70.4, 111.6 (2C), 121.4, 121.9, 124.5, 126.3, 129.2, 131.9 synthesized and purified as described above for 9, starting
(
2C), 151.4, 164.2.
from compound 14 (700 mg, 1.63 mmol). Yield: 36% (303 mg,
1
6
-Methyl-(2-(N-methyl-N-(2′-(4′-bromobutoxy)ethyl)-4′-amino- 0.59 mmol); R = 0.41 (CHCl –MeOH 97 : 3); H NMR (CDCl ,
f
3
3
phenyl))-benzothiazole (12). Compound 12 was synthesized 300 MHz): δ 1.23 (t, 3H, J = 7.2 Hz), 1.60 (m, 4H), 2.42 (s, 3H),
and purified as described above for 11, starting from com- 2.49 (m, 2H), 2.61 (t, 2H, J = 6.3 Hz), 2.76 (t, 2H, J = 6.3 Hz),
pound 4 (1.0 g, 3.35 mmol). Yield: 40% (581 mg, 1.34 mmol); 3.02 (s, 3H), 3.39 (m, 4H), 3.55 (s, 4H), 4.14 (q, 2H, J = 7.2 Hz),
1
3
3
R
f
= 0.64 (n-hexane–AcOEt 1 : 1); H NMR (CDCl
3
, 300 MHz): 6.70 (d, 2H, J = 9.0 Hz), 7.20 (d, 1H, J = 8.4 Hz), 7.58 (s, 1H),
3
3
13
δ 1.64 (m, 2H), 1.86 (m, 2H), 2.42 (s, 3H), 3.00 (s, 3H), 3.37 (m, 7.82 (d, 1H, J = 8.1 Hz), 7.88 (d, 2H, J = 9.0 Hz); C NMR
3
3
4
8
8
3
H), 3.53 (s, 4H), 6.69 (d, 2H, J = 8.7 Hz), 7.2 (d, 1H, J = (CDCl , 300 MHz): δ 13.2, 20.5, 25.2, 27.8, 30.2, 30.7, 38.1,
3
3
3
.2 Hz), 7.57 (s, 1H), 7.82 (d, 1H, J = 8.2 Hz), 7.87 (d, 2H, J = 46.9, 48.8, 51.2, 60.3, 67.2, 69.8, 110.6 (2C), 120.2, 120.5, 120.8,
1
3
.7 Hz); C NMR (CDCl
3
, 75 MHz): δ 21.4, 28.1, 29.5, 33.7, 126.5, 127.8 (2C), 133.2, 133.7, 150.0, 151.5, 166.7; ESI/MS
9.0, 52.0, 70.2, 111.4 (2C), 121.1, 121.3, 121.6, 127.3, 128.7 C H N O S (515.2) m/z 516.3 [M + H] .
+
2
7
37 3 3 2
(
(
2C), 134.1, 134.5, 150.8, 152.3, 167.6; ESI/MS C21
433) m/z 434.4 [M + H] .
H
25BrN
2
OS
2-[(2-((2″-((4″-(Benzo[d]thiazol-2-yl)phenyl)(methyl)amino)-
ethyl)thio]ethyl)amino) acetic acid (L1). A solution of 9
+
2-[N-Methyl-N-(2′-(4″-(2′′′-aminoethylthio)butoxy)ethyl)-4′-amino- (148 mg, 0.35 mmol) in THF (10 mL) and water (15 mL) was
phenyl]-benzothiazole (13). Compound 13 was synthesized and refluxed with NaOH (136 mg, 3.40 mmol) for 17 h. After
purified as described above for 7, starting from compound 11 cooling down, the reaction mixture was neutralized with 1 M
(
(
1.2 g, 2.86 mmol). Yield: 46% (547 mg, 1.32 mmol); R = 0.42 HCl, followed by extraction with CHCl3 (2 × 25 mL). The
f
1
CHCl
3
–MeO 4 : 1); H NMR (CD
3 2 4
OD, 300 MHz): δ 1.62 (m, organic phase was dried over Na SO , filtered and the filtrate
4
3
H), 2.51 (m, 2H), 2.63 (t, 2H, J = 6.6 Hz), 2.9 (t, 2H, J = 6.6 Hz), was concentrated. Recrystallization of the precipitate in metha-
3
.08 (s, 3H), 3.46 (m, 2H), 3.64 (s, 4H), 6.73 (d, 2H, J = 9.0 Hz), nol afforded L1 as a white solid. Yield: 58% (80 mg,
3
3
3
1
7
f 3
.28 (dd, 1H, J = 7.5 Hz, J = 7.8 Hz), 7.41 (dd, 1H, J = 7.9 Hz, 0.20 mmol); R = 0.29 (CHCl –MeOH 3 : 1); H NMR (DMSO,
3
3
3
J = 7.5 Hz), 7.82 (d,1H, J = 7.9 Hz), 7.92 (d, 2H, J = 9.0 Hz), 300 MHz): δ 2.80 (m, 4H), 2.98 (t, 2H, J = 8.1 Hz), 3.03 (s, 3H),
3
13
3
7
2
1
.96 (d, 1H, J = 7.8 Hz); C NMR (CD OD, 75 MHz): δ 27.3, 3.21 (s, 2H), 3.62 (t, 2H, J = 8.1 Hz), 6.84 (d, 2H, J = 9.0 Hz),
9.8, 32.2, 39.3, 40.5, 52.9, 69.5, 71.7, 112.9 (2C), 122.6, 122.6, 7.39 (ddd, 1H, J = 1.2 Hz, J = 7.8 Hz, J = 7.5 Hz), 7.51 (ddd,
22.7, 125.7, 127.4, 129.9 (2C), 135.3, 153.2, 155.1, 171.1.
3
4
3
3
4
3
3
3
1H, J = 1.2 Hz, J = 7.5 Hz, J = 8.1 Hz), 7.89 (d, 2H, J = 9.0
3
3
13
6
-Methyl-2-[N-methyl-N-(2′-(4″-(2′′′-aminoethylthio)butoxy)- Hz), 7.92 (d, 1H, J = 8.1 Hz), 8.04 (d, 1H, J = 7.8 Hz); C NMR
ethyl)-4′-aminophenyl]-benzothiazole (14). Compound 14 (DMSO, 75 MHz): δ 25.7, 46.4, 49.6, 51.3, 56.1, 111.8 (2C),
was synthesized and purified as described above for 7, starting 120.3, 122.0, 124.5, 126.4, 128.7 (2C), 133.8, 150.7, 153.9,
+
from compound 12 (900 mg, 2.08 mmol). Yield: 86% (767 mg, 167.8; ESI/MS C H N O S (401.55) m/z 402.2 [M + H] , 424.2
2
0
23 3 2 2
1
+
1
3
2
2
1
2
3
1
.79 mmol); R
00 MHz): δ 1.56 (m, 4H), 2.39 (s, 3H), 2.44 (m, 2H), 2.59 (t, 6.09, N 9.86; found C 56.54, H 6.15, N 9.61.
H, J = 6.4 Hz), 2.86 (t, 2H, J = 6.4 Hz), 2.97 (s, 3H), 3.35 (m, 2-[2′-(2″-((4′′′-(6″″-Methylbenzo[d]thiazol-2-yl)phenyl)(methyl)-
H), 3.51 (s, 4H), 3.76 (s, 2H), 6.67 (d, 2H, J = 8.8 Hz), 7.17 (d, amino)ethylthio)ethylamino] acetic acid (L2). L2 was syn-
f
= 0.58 (CHCl
3
–MeOH 3 : 1); H NMR (CDCl
3
,
23 3 2 2 2
[M + Na] ; Anal. calcd for C20H N O S ·1.4H O: C 56.38, H
3
3
3
H, J = 8.4 Hz), 7.54 (s, 1H), 7.80 (d, 1H, J = 8.4 Hz), 7.85 (d, thesized and purified as described above for L1, starting from
H, J = 8.8 Hz); C NMR (CDCl , 75 MHz): δ 21.3, 26.1, 28.6, compound 10 (30 mg, 0.067 mmol). Yield: 65% (18 mg,
1.4, 34.0, 38.8, 40.3, 52.0, 68.0, 70.6, 111.4 (2C), 121.0, 121.2, 0.04 mmol); H NMR (CD OD, 300 MHz): δ 2.46 (s, 3H), 3.09
3
21.5, 127.3, 128.6 (2C), 134.0; 134.4, 150.8, 152.3, 167.5; ESI/ (s, 2H), 2.85 (m, 4H), 3.42 (s, 3H), 3.69 (m, 4H), 6.84 (d, 2H,
3
13
3
1
+
3
3
MS C H N OS (429.2) m/z 430.3 [M + H] .
J = 9.0 Hz), 7.28 (d, 1H, J = 8.4 Hz), 7.70 (s, 1H), 7.76 (d, 1H,
J = 8.4 Hz), 7.87 (d, 2H, J = 9.3 Hz); C NMR (CD OD,
3
23
31
3
2
3
3
13
Ethyl 2-[4′-(benzo[d]thiazol-2-yl)phenyl]-5-oxa-10-thia-2,13-
diazapentadecan-15-oate (15). Compound 15 was synthesized 75 MHz): δ 22.5, 25.3, 28.3, 29.2, 37.8, 67.7, 111.7 (2C), 121.0,
and purified as described above for 9, starting from compound 121.2, 127.7, 128.7 (2C), 134.9; ESI/MS C H N O S (415.14)
2
1
25 3 2 2
+
+
1
0
3 (500 mg, 1.20 mmol). Yield: 66% (397 mg, 0.79 mmol); R
f
=
m/z 416.3 [M + H] , 438.3 [M + Na] ; Anal. calcd for
·1.6H O: C 56.75, H 6.12, N 9.46; found C 55.98,
1
.53 (CHCl
3
–MeOH 95 : 5); H NMR (CDCl
3
, 300 MHz): δ 1.23
C
21
H
25
N
3
O
2
S
2
2
(t, 3H, J = 7.2 Hz), 1.61 (m, 4H), 2.50 (m, 2H), 2.62 (t, 2H, J = H 6.48, N 9.27.
6
.2 Hz), 2.77 (t, 2H, J = 6.2 Hz), 3.04 (s, 3H), 3.40 (m, 4H), 3.57
[4-(Benzo[d]thiazol-2-yl)phenyl]-5-oxa-10-thia-2,13-diazapen-
3
(
(
s, 4H), 4.15 (q, 2H, J = 7.2 Hz), 6.72 (d, 2H, J = 9.0 Hz), 7.26 tadecan-15-oic acid (L3). L3 was synthesized and purified as
dd, 1H, J = 7.2 Hz, J = 7.8 Hz), 7.40 (dd, 1H, J = 7.2 Hz, J = described above for L1, starting from compound 15 (350 mg,
3
3
3
3
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Paper
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1
+
0
.70 mmol). Yield: 52% (172 mg, 0.36 mmol); H NMR
C
24
H
24
N
3
O
5
S
2
Re (685.07) (m/z) 686.3 [M + H] (100); Anal.
(
(
(
CD OD, 300 MHz): δ 1.63 (m, 4H), 2.55 (t, 2H, J = 7.2 Hz), 2.75 calcd for C H N O S Re: C 42.09, H 3.53, N 6.14; found C
3 24 24 3 5 2
t, 2H, J = 7.2 Hz), 3.08 (s, 3H), 3.14 (t, 2H, J = 6.9 Hz), 3.47 42.25, H 3.75, N 6.09.
3
1
m, 4H), 3.64 (s, 4H), 6.86 (d, 2H, J = 7.2 Hz), 7.34 (ddd, 1H,
Re3 was obtained in 76% yield (78 mg, 0.11 mmol);
H
3
4
3
J = 7.8, 7.5 Hz, J = 1.2, 0.6 Hz), 7.46 (td, 1H, J = 7.8; 7.5 Hz, NMR (DMF-d , 300 MHz, T = 20 °C): δ 1.71 (m, 4H), 3.08 (s,
7
4
13
3
J = 1.2; 0.6 Hz), 7.89 (m, 4H); C NMR (CD OD, 75 MHz): 4H), 3.50 (s, 3H), 3.65 (m, 5H), 6.90 (d, 2H, J = 8.4 Hz), 7.39 (m,
1
3
δ 27.1, 28.6, 29.8, 32.1, 39.3, 52.9, 66.9, 69.5, 71.7, 112.9 (2C), 1H), 7.50 (m, 1H), 7.95 (m, 3H); C NMR (DMF-d
1
1
7
, 300 MHz,
21.6, 122.6, 122.7, 125.7, 127.4, 129.9 (2C), 135.3, 153.2, T = 20 °C): δ 25.01, 28.32, 37.10, 38.54, 51.53, 53.74, 55.76,
55.1, 171.1 (CvO); ESI/MS C24 (473.18) m/z 472.2 68.13, 70.10, 111.90, 120.18, 121.82, 121.97, 124.78, 126.56,
H
31
N
3
O
3
S
2
+
[M − H] ; Anal. calcd for C24
H
31
N
3
O
3
S
2
·2.2H
2
O: C 56.16, H 128.79, 134.10, 151.86, 154.14, 168.93, 181.51, 192.31, 196.12,
6
.95, N 8.19; found C 56.63, H 7.09, N 8.02.
197.75; IR (KBr) ν: 4008, 2923, 2027, 1896, 1748, 1606, 1559,
2
-[4′-((6″-Methyl)benzo[d]thiazol-2-yl)phenyl]-5-oxa-10-thia- 1541, 1508, 1487, 1456, 1384, 1350, 1261, 1183, 1101, 816, 691,
,13-diazapentadecan-15-oic acid (L4). L4 was synthesized 651, 567, 516 cm− ; ESI/MS (+) C27
3
H
N
O
S
Re (743.0) (m/z)
2
30
3
+
6
2
+
and purified as described above for L1, starting from com- 744.4 [M
pound 16 (250 mg, 0.48 mmol). Yield: 65% (154 mg,
+
H] , 766.4 [M
+
Na] ; Anal. calcd for
C
27
H
30
N
3
O
5
S
2
Re: C 43.65, H 4.07, N 5.66; found C 43.48, H
1
0
(
2
.32 mmol); H NMR (CD
3
OD, 300 MHz): δ 1.60 (m, 4H), 2.52 4.24, N 5.54.
Re4 was obtained in 81% yield (86 mg, 0.11 mmol);
H, J = 7.2 Hz), 3.45 (s, 3H), 3.62 (s, 4H), 6.80 (d, 2H, J = 9.0 NMR (DMF-d
1
t, 2H, J = 7.2 Hz), 2.74 (t, 2H, J = 7.2 Hz), 3.06 (s, 2H), 3.12 (t,
H
3
7
, 400 MHz, T = 20 °C): 1.74 (s, 2H), 1.83 (s, 2H),
3
3
Hz), 7.24 (d, 1H, J = 8.4 Hz), 7.68 (s, 1H), 7.72 (d, 1H, J = 8.4 2.47 (s, 3H), 3.12 (s, 5H), 3.41 (q, 2H, J = 3.2 Hz), 3.52 (s, 2H),
Hz), 7.82 (d, 2H, J = 9.0 Hz); C NMR (CD OD, 75 MHz): 3.68 (s, 5H), 6.93 (d, 2H, J = 7.4 Hz), 7.33 (s, 1H), 7.85 (m, 2H),
δ 27.5, 28.9, 30.1, 32.5, 39.7, 53.4, 67.3, 69.9, 72.1, 112 (2C), 7.94 (d, 2H, J = 7.4 Hz); C NMR (DMF-d
3
13
3
1
3
7
, 400 MHz, T =
1
23.0, 123.2, 129.8, 129.0 (2C), 135.2.2, 153.4, 155.3, 171.4 20 °C): δ 15.94, 21.68. 26.28, 35.02, 35.23, 37.86, 39.30, 39.59,
+
(CvO); ESI/MS C H N O S (487.2) m/z 488.4 [M + H] , 510.4 52.69, 54.80, 56.08, 66.38, 69.19, 71.13, 112.90, 121.80, 122.65,
25 33 3 3 2
+
[M + Na] . Anal. calcd for C25
H
33
N
3
O
3
S
2
·1.5H
2
O: C 58.34, H 128.79, 129.61, 135.52, 152.61, 153.55, 168.26, 171.92, 177.35,
6
.75, N 8.17; found C 57.24, H 6.88, N 8.24.
180.44, 194.18, 197.49; IR (KBr) ν: 3490, 2920, 2028, 1895,
−
3
1
653, 1606, 1558, 1506, 1457, 1384, 1261, 1100, 921, 688 cm ;
General procedure for the synthesis of the rhenium complexes
Re1–Re4)
fac-[Re(H O) (CO) ]Br was reacted with equimolar amounts of H 4.26, N 5.55; found C 43.97, H 4.6, N 5.43.
+
ESI/MS (+) C28
H
32
N
3
O
6
S
2
Re (757.0) (m/z) 758.6 [M + H] (100),
(
+
7
80.5 [M + Na] (80); Anal. calcd for C28 Re: C 44.43,
32 3 6 2
H N O S
2
3
3
L1–L4 in refluxing methanol for 16 h. Thereafter, the solvent
was removed under vacuum and the desired complexes were
purified by successive washing with water, diethyl ether and
9
9m
General procedure for the synthesis of
Tc complexes
n-hexane. The obtained yellow solids were further recrystal- In a nitrogen-purged glass vial, 120 μL of a 1.1 × 10− M solu-
3
lized from methanol to afford pure samples of Re1–Re4, as tion of L1–L4 in propanediol-1,3 was added to 1.2 mL of a
9
9m
checked by HPLC analysis.
solution of the organometallic precursor fac-[ Tc-
1
+
Re1 was obtained in 62% yield (54 mg, 0.08 mmol);
H
2 3 3
(H O) (CO) ] (1–2 mCi) in saline at pH 7.4. The reaction mix-
NMR (DMF-d
7
, 300 MHz, T = 20 °C): 2.64 (m, 2H), 3.20 (s, 4H), tures were then heated to 100 °C for 30 min, cooled to room
3
.38 (m, 2H), 3.71 (m, 2H), 3.96 (m, 2H), 7.05 (d, 2H, J = 9.0 temperature and analyzed by RP-HPLC, using the method
9
9m
Hz), 7.40 (m, 1H), 7.52 (m, 1H), 7.98 (m, 3H), 8.10 (d, 1H, J = described above. Following this procedure, all
Tc complexes
1
3
7
5
1
1
1
7
.8 Hz); C NMR (DMF-d , 300 MHz, T = 20 °C): δ 39.1, 51.50, (Tc1–Tc4) were synthesized in radiochemical yields >95%
3.32, 54.40, 55.43, 56.40, 113.09, 113.47, 122.93, 123.27, without the need for further purification. Their chemical iden-
25.64, 127.40, 129.95, 135.60, 152.30, 155.71, 169.13, 180.16, tity was ascertained by HPLC comparison with the Re conge-
93.72, 197.17; IR (KBr) ν: 3410, 2922, 1894, 1699, 1606, 1486, ners (see Table 1).
−
3
455, 1384, 1261, 1191, 1104, 817 cm
;
ESI/MS (+)
+
C
23
H
22
N
3
O
5
S
2
Re (671.0) (m/z): 672.2 [M + H] (100); Anal. calcd
Determination of partition coefficients
for C23
H
22
N
3
O
5
S
2
Re: C 41.18, H 3.31, N 6.27; found C 41.07, H
3
.47, N 6.19.
Re2 was obtained in 57% yield (32 mg, 0.05 mmol);
The log Do/w values of complexes Tc1–Tc2 were determined by
the “shake flask” method. A mixture of octanol (1 mL) and
1
28
H
7
NMR (DMF-d , 300 MHz, T = 20 °C): δ 2.47 (m, 6H), 3.19 (s, 0.1 M PBS pH = 7.4 (1 mL) was stirred vigorously, followed by
1
3
9
H), 6.99 (s, 2H), 7.32 (s, 1H), 7.83 (m, 2H), 7.95 (m, 2H);
C
the addition of 25 μL of the aqueous solutions of each
NMR (DMF-d
7
, 300 MHz, T = 20 °C): δ 21.18, 32.33, 38.51, complex. The mixtures were vortexed and centrifuged
3
1
1
8.66, 47.99, 50.39, 50.83, 52.65, 54.30, 54.95, 55.66, 55.96, (3000 rpm, 10 min, RT) to allow phase separation. Aliquots of
12.36, 112.72, 121.41, 122.09, 128.35, 129.17, 134.90, 135.24, 25 μL of the octanol and PBS phases were counted in a gamma
51.32, 152.88, 167.69, 181.17, 192.76, 196.56, 198.16; IR (KBr) counter. The partition coefficient (Po/w) was calculated by
ν: 3395, 2924, 2028, 1893, 1700, 1604, 1509, 1483, 1437, 1384, dividing the number of counts of the octanol phase by those
−
3
1
261, 1193, 1108, 817, 759, 698, 650 cm ; ESI/MS (+) from the PBS phase, and the results are expressed as log Do/w.
5192 | Org. Biomol. Chem., 2015, 13, 5182–5194
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Paper
Cell culture
Cellular uptake
9
9m
The human MCF7 breast and PC3 prostate cells (American Cellular uptake assays of the
Tc complexes (Tc1–Tc4) were
Type Culture Collection, ATCC) were grown respectively in performed using PC-3 and MCF-7 cells seeded at a density of
4
DMEM and RPMI media, supplemented with 10% (v/v) fetal 4 × 10 cells in 500 μL medium per well in 24-well plates and
bovine serum and 1% penicillin–streptomycin (all from Invi- allowed to attach overnight. After that period, the medium was
trogen). Cells were cultured at 37 °C in a 5% CO
Heraus, Germany) under a humidified atmosphere, with the approximately 25 kBq mL− of each
medium changed every other day. bated under a humidified 5% CO atmosphere, at 37 °C for a
2
incubator removed and cells were treated with fresh medium containing
1
99m
(
Tc complex and incu-
2
period of 30 min to 18 h. Cells were washed twice with cold
PBS, lysed with 0.1 M NaOH and the cellular extracts were ana-
lyzed for radioactivity. Each experiment was performed in
duplicate with each point determined in at least four repli-
cates. Cellular uptake data were expressed as an average plus
the standard deviation of % of total per million of cells.
Cytotoxicity assays
The cell viability was evaluated by using a colorimetric method
based on the tetrazolium salt MTT ([3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide]), which is reduced by
living cells to yield purple formazan crystals. Cells were seeded
4
Biodistribution studies
in 96-well plates at a density of 1–1.5 × 10 cells per well in
2
00 μL of culture medium and incubated overnight. After
All animal experiments were performed in compliance with
Portuguese regulations for animal treatment. The animals
were housed in a temperature- and humidity-controlled room
with a 12 h light/12 h dark schedule. Biodistribution of com-
plexes Tc1–Tc4 was evaluated in CD-1 mice (randomly bred,
obtained from IFFA, CREDO, Spain) weighing approximately
careful removal of the medium, 200 μL of a dilution series of
the compounds in fresh medium were added and incubation
was performed at 37 °C/5% CO for 72 h. The percentage of
2
DMSO in cell culture medium did not exceed 1%. At the end
of the incubation period, the compounds were removed and
the cells were incubated with 200 μL of MTT solution (500 µg
ml ). After 3 h, the medium was removed and the purple for-
mazan crystals were dissolved in 200 μL of DMSO by shaking.
The cell viability was evaluated by the measurement of the
absorbance at 570 nm using a plate spectrophotometer (Power
Wave Xs, Bio-Tek). The cell viability was calculated by the ratio
between the absorbance of each well and that of the control
wells (cells treated with medium containing 1% DMSO). Each
experiment was repeated at least three times and each point
was determined in at least six replicates. Data were analyzed
with GraphPad Prism software.
20–25 g. Animals were intravenously injected in the tail vein
−
1
with the test complex (1.8–7.8 MBq) diluted in 100 μL of NaCl
0
1
.9%. Mice were sacrificed by cervical dislocation at 2 min and
h after injection. The administered dose and the radioactivity
in the sacrificed animals were measured using a dose calibra-
tor (Curiemeter IGC-3, Aloka, Tokyo, Japan). The difference
between the radioactivity in the injected and sacrificed
animals was assumed to be due to excretion. The tissues of
interest were dissected, rinsed to remove excess blood,
weighed, and their radioactivity was measured using a
γ-counter (LB2111, Berthold, Germany). The uptake in the
tissues was calculated and expressed as a percentage of the
injected radioactivity dose per gram of tissue.
Fluorescence spectra and fluorescence microscopy analysis
The excitation and emission wavelengths of L3 and Re3 (25 μM In vivo stability studies
in PBS) were recorded using a plate reader (Tecan Infinite 200,
The in vivo stability of Tc1–Tc4 was evaluated by urine and
blood serum HPLC analysis, using the elution conditions
Männedorf, Switzerland). The maximum excitation and emis-
sion wavelengths were determined according to the maximum
fluorescence intensity level.
99m
above described for the analysis of these
urine was collected at the sacrifice time and centrifuged for
min at 1500g before RP-HPLC analysis. Blood collected from
Tc complexes. The
MCF7 and PC3 cells, cultured in 35 mm imaging dishes
5
(Ibidi), were treated with a vehicle (DMSO) or with the ligands
mice was also centrifuged for 10 min at 1000g at 4 °C, and the
supernatant (serum) was collected. Aliquots of 100 μL of
serum were treated with 200 μL of cold ethanol for protein pre-
cipitation. Samples were centrifuged at 1500g for an additional
L3 or the complex Re3 (100 μM), for 24 h. Cells washed with
PBS were fixed and permeabilized in 100% ice-cold methanol
(
−20 °C, 5 min). Cells, washed with PBS 3 times, were incu-
−1
bated with 20 μg ml propidium iodide (nuclear staining) for
min at RT. Cells were washed with PBS 3 times and were kept
1
0 min, at 4 °C. The remaining supernatant was separated and
injected through a RP-HPLC column (Nucleosil 100-10, 250 ×
mm) for analysis.
4
at 4 °C until analysis. Samples were imaged on a widefield
fluorescence microscope Zeiss Axiovert 200M (Carl Zeiss
MicroImaging) using a 63× Plan-Apochromat using the Red
3
(
3
λ
ex = 540–552 nm, λem = 575–640 nm) and Blue (λex
=
Acknowledgements
59–371 nm, λem > 397 nm) filter sets. Images were acquired
using a cooled CCD camera (Roper Scientific Coolsnap HQ This work was funded by the Fundação para a Ciência e Tecno-
CCD) using Zeiss Metamorph software. All images were pro- logia (FCT), Portugal (EXCL/QEQ-MED/0233/2012). The
2
9
cessed using ImageJ – Image Processing and Analysis in Java.
research was carried out within the framework of the European
This journal is © The Royal Society of Chemistry 2015
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Cooperation COST Action CM1105. FCT is also acknowledged 11 C. G. Mortimer, G. Wells, J.-P. Crochard, E. L. Stone,
for “Ciência 2008” program (GRM), FCT Investigator Grant
T. D. Bradshaw, M. F. G. Stevens and A. D. Westwell, J. Med.
Chem., 2006, 49, 179.
(FM) and grants SFRH/BPD/80758/2011 (EP), SFRH/BD/47308/
2008 (FS) and SFRH/BPD/64702/2009 (HVM). TFO is supported 12 A. I. Loaiza-Pérez, V. Trapani, C. Hose, S. S. Singh,
by the DFG Center for Nanoscale Microscopy and Molecular
Physiology of the Brain (CNMPB). The authors thank Dr
J. B. Trepel, M. F. G. Stevens, T. D. Bradshaw and
E. A. Sausville, Mol. Pharmacol., 2002, 61, 13.
Joaquim Marçalo and Dr Célia Fernandes for Mass Spec- 13 V. Trapani, V. Patel, C.-O. Leong, H. P. Ciolino,
troscopy analysis, which was carried out on a QITMS instru-
ment, acquired with the support of the Programa Nacional de
Reequipamento Científico (Contract REDE/1503/REM/2005-
G. C. Yeh, C. Hose, J. B. Trepel, M. F. G. Stevens,
E. A. Sausville and A. I. Loaiza-Pérez, Br. J. Cancer, 2003,
88, 599–605.
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