Novel heterobimetallic radiotheranostic: Preparation, activity, and biodistribution

Article abstract of DOI:10.1002/cmdc.201300494

A novel Ru^II(arene) theranostic complex is presented. It is based on a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid macrocycle bearing a triarylphosphine and can be tracked in vivo by using the γ emission of ¹⁵³Sm atoms.

Full text of DOI:10.1002/cmdc.201300494

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DOI: 10.1002/cmdc.201300494
Novel Heterobimetallic Radiotheranostic: Preparation,
Activity, and Biodistribution
Louis Adriaenssens,[a] Qiang Liu,[a] Fanny Chaux-Picquet,[a] Semra Tasan,[a] Michel Picquet,[a]
Franck Denat,[a] Pierre Le Gendre,[a] Fernanda Marques,[b] Cꢀlia Fernandes,[b] Filipa Mendes,[b]
Lurdes Gano,[b] Maria Paula Cabral Campello,*[b] and Ewen Bodio*[a]
A novel RuII(arene) theranostic complex is presented. It is
based on a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid macrocycle bearing a triarylphosphine and can be tracked
in vivo by using the g emission of 153Sm atoms. Notably, the
heteroditopic ligand can be selectively metalated with rutheni-
um at the phosphorus atom despite the presence of other
functionalities that are prone to metal coordination. Subse-
quent labeling with radionuclides such as 153Sm can then be
performed easily. The resulting heterobimetallic complex ex-
hibits favorable solubility and stability properties in biologically
relevant media. It also shows in vitro cytotoxicity in line with
that expected for this type of metallodrug, and is nontoxic to
the organism as a whole. As a proof of concept, initial studies
in healthy mice were performed to obtain information about
the uptake, biodistribution, and excretion of the radiolabeled
complex.
Introduction
Metallic complexes have been at the forefront of cancer thera-
py for decades. Indeed, cisplatin and its derivatives are some
of the most widely used anticancer drugs in the world, specifi-
cally against testicular and ovarian cancers.[1] This success has
raised the profile of bioinorganic chemistry, which has, in
recent years, seen the development of a plethora of metal
complexes targeted toward therapeutic applications.[2] An as-
sortment of complexes with different metals have proven to
be active in a variety of biologically relevant assays, with the
standout metals being platinum and ruthenium. In particular,
the Ru(arene) complexes reported by Dyson, Sadler, and their
respective co-workers have shown remarkable anticancer or
anti-metastatic activities (complexes I and II; Figure 1).[3]
With the exception of cisplatin and related species, the ma-
jority of early biometallic (bioinorganic and bioorganometallic)
research was centered on the discovery of complexes that dis-
played maximum activity. Such an approach led to the discov-
ery of several very potent biometallics but meant that the
thoughtful modification of these species was challenging. In
the last five years, a shift has taken place in the field, with
Figure 1. Examples of known ruthenium-based complexes with very promis-
ing anticancer activity.
more emphasis being placed on understanding how these
metallodrugs work. In this respect, theranostics have contribut-
ed richly to our understanding. Initially, theranostics, a neolo-
gism built from the contraction of the words therapy and diag-
nostic, were compounds that helped in the identification of
a patient’s disease in order to select the specific therapy re-
quired (this method was previously called personalized medi-
cine). Today, chemists enlarge this field to compounds com-
posed by both a therapeutic moiety and an imaging agent.
This specificity allows researchers to follow the theranostic
during its “life”, which gives some clues on its mechanism of
action. Clearly, in an investigation of the mechanism of action,
virtually the entirety of all cellular components (proteins, DNA,
RNA, etc.) in the whole body must be considered. This level of
complexity seriously hampers our ability to fully comprehend
how therapeutic metallic complexes work and, thus, it also
hampers our attempts to make new, more efficient metallo-
drugs.[3e,4]
[a] Dr. L. Adriaenssens, Dr. Q. Liu, Dr. F. Chaux-Picquet, Dr. S. Tasan,
Dr. M. Picquet, Prof. F. Denat, Prof. P. Le Gendre, Dr. E. Bodio
Institut de Chimie Molꢀculaire de l’Universitꢀ de Bourgogne
UMR 6302 CNRS, Universitꢀ de Bourgogne
9 avenue A. Savary, BP47870, 21078 Dijon (France)
E-mail: ewen.bodio@u-bourgogne.fr
[b] Dr. F. Marques, Dr. C. Fernandes, Dr. F. Mendes, Dr. L. Gano,
Dr. M. P. C. Campello
Centro de CiÞncias e Tecnologias Nucleares
Instituto Superior Tꢀcnico, Universidade de Lisboa
Estrada Nacional 10, km 139.7, 2695-066 Bobadela LRS (Portugal)
E-mail: pcampelo@ctn.ist.utl.pt
Scintigraphic imaging of radionuclides has proven to be
a valuable tool to gain a big-picture idea of what’s happening
in the body. It occurred to us that a ligand suitable for the si-
multaneous coordination of therapeutically relevant metals
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300494.
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and radionuclides would allow the localization of the corre-
sponding relevant metal complexes within the body. Chiefly,
our major goal was to reveal the organ specificity, the uptake
rate, and the clearance of these fascinating complexes.
agent. Indeed, this was the case and among several envisioned
strategies, only one allowed access to the desired product in
satisfactory yield.
To minimize the synthetic complications, we coupled the
phosphine ligand and the DOTA macrocycle at the last step of
the synthesis of bifunctional heterotopic ligand 1. This conver-
gent strategy entailed an amide coupling between a DOTA
macrocycle decorated with an amine arm, 2, and the phos-
phine activated ester 3 (Scheme 1a). Both coupling partners
Thus, we selected heterotopic ligand 1 (Figure 2). Phosphine
complexes are among the most promising biometallics and,
Figure 2. Proposed strategy for the heterobimetallic radiotheranostic agents.
therefore, the ligand is based on a functionalized triphenyl-
phosphine moiety.[5] In this manner, we hoped to produce Ru–
phosphine complexes that share a resemblance in nature and,
hopefully, in activity with known potential metallodrugs. On
the other part of the ligand, we envisaged a 1,4,7,10-tetraaza-
cyclododecane-1,4,7,10-tetraacetic acid (DOTA) moiety, which
would be used to complex the radionuclide. DOTA-derivative
macrocycles have proven to be excellent ligands for a variety
of radionuclides, such as 111InIII, 68GaIII, and 153SmIII atoms, all of
which have been used for imaging and/or therapy.[6] Similarly
to the 1,3,5-triaza-7-phosphaadamantane (PTA) ligand used by
Dyson and co-workers to construct RAPTA complexes, DOTA
macrocycles and their metal complexes are known to be
highly water soluble, and so we envisaged that the covalent
attachment of such a moiety would impart a favorable solubili-
ty profile in biologically relevant media. In addition, we,
among others, have successfully developed modified DOTA
macrocycles that act as bifunctional chelating agents, and we
were therefore optimistic that DOTA macrocycles could be
ideal candidates to create the proposed heteronuclear com-
plex.[7]
Scheme 1. a) Amide coupling to produce heteroditopic ligand 1. Reagents
and conditions: DIPEA, MeCN, RT, overnight. b) 1H and 31P{1H} NMR spectra
(in [D6]DMSO) at 300 K (top) and 400 K (bottom) of 1; diagnostic protons are
labeled.
were prepared by routes previously used in our laboratories.[8]
These conditions provided the desired heterotopic ligand 1 in
57% yield and in sufficient quantities for the metalation stud-
1
ies and biological tests. The H NMR spectrum of heteroditopic
ligand 1 displayed broad and poorly defined signals and was
initially difficult to interpret. Such behavior is typical for DOTA
derivatives and often hampers confident characterization.
However, upon heating the sample to 400 K, the spectrum
became well-resolved, and the key proton signals could be
clearly observed and assigned (Scheme 1b). The 31P{1H} NMR
spectrum displayed a single signal in the expected region at
d=ꢀ5.7 ppm.
Results and Discussion
Synthesis of the heterotopic ligand
The chemistry involved in the preparation of complexes resem-
bling RAPTA is relatively sensitive, especially during and after
rutheniation. Conversely, the chemistry of DOTA-like macrocy-
cles can be broadly thought of as amino acid chemistry and
appears, at first glance, to be incompatible with the delicate
nature of RuII complexes. Thus, we anticipated synthetic diffi-
culties in obtaining the targeted heterobimetallic theranostic
Selective complexation
The main synthetic challenge of this work was to succeed in
selectively coordinating the Ru atom at the phosphine moiety
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thanesulfonate in acetonitrile at
room temperature (Scheme 3).
Overnight, the reaction came
to completion and produced
the envisaged heterobimetallic
theranostic agent 5.
Radiolabeling
After the successful production
of complex 5, we proceeded
with the radiochemical labeling
studies. By using HPLC analysis,
the reaction kinetics of 153Sm
with 4 was found to be depen-
dent on temperature, pH value,
and the metal/ligand molar ratio
(M/L). We initially performed the
labeling at room temperature
with a 1:2 metal/ligand molar
ratio and pH 5.5. Under these
Scheme 2. Selective rutheniation of the phosphine moiety of heteroditopic ligand 1. Insets on the right:
31P{1H} NMR (121.5 MHz, 300 K, CDCl3) spectra of the free ligand 1 (top) and the ruthenium complex 4 (bottom).
conditions, poor labeling yields
were obtained, even after a 24 h
reaction time. However, after
only 10 min at 708C, the labeling
of 1 and then in chelating the radionuclide within the attached
DOTA cage without disturbing the phosphine–metal complex.
Due to the practicalities of handling radioactive compounds
and the short half-life of the radiometal, the chelation of the
radionuclide must occur just before administration and is,
thus, essentially the last step. With this in mind, we were
pleased to find that rutheniation of 1 occurred solely at the
phosphorus atom and could be achieved in good yield by
simply adding the metal precursor to a solution of
heteroditopic ligand 1 (Scheme 2). Despite the pref-
erence of ruthenium for the soft phosphorus atom,
the selectivity of the metalation was not entirely ex-
pected. Given the sheer quantity of functionality dis-
played by the DOTA moiety, which is known to coor-
dinate ruthenium, it is notable that the metal can dis-
criminate between the phosphine and the tetraaza-
macrocycle. In the event, 0.5 equivalent of [RuCl2(p-
cymene)]2 was added to a solution of heteroditopic
ligand 1 in chloroform. Simple precipitation followed
by solvent washing provided the pure complex 4.
The complexation was monitored by 31P{1H} NMR
spectroscopy (Scheme 2, inset on the right). A signifi-
cant shift of the resonance was observed upon going
from the free ligand (singlet at d=ꢀ5.7 ppm) to the
complex (singlet at d=24.9 ppm).
yield was high, no colloidal species were found by ITLC-SG
analysis, and no free 153Sm species were detected in the HPLC
chromatogram (tR =2.5 min). As can be seen in Figure 3, one
major radiochemical species (tR =12.5 min) representing ~85%
of the total activity and two minor peaks (tR =11.3 and
13.4 min) were obtained. The major peak corresponds to the
radiocomplex [153Sm]5 and its authenticity was established by
comparing its chromatographic behavior with that of the inac-
Initial metalation studies of the DOTA macrocycle
were performed with cold samarium. This was done
to obtain material for cytotoxicity evaluation and, im-
portantly, to identify and characterize the analogous
radioactive compound [153Sm]5. The best results were
Scheme 3. a) Metalation of the DOTA macrocycle under mild conditions gives theranostic
agent 5. b) The observed high-resolution mass spectrum (left) matches perfectly with the
obtained by simply stirring the DOTA–ruthenium-
(arene) adduct with 1.2 equiv samarium trifluorome- calculated spectrum (right) to confirm the identity of 5.
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could not be said for ligand 1. All of the mice treated with
1 died more or less immediately after administration. This tox-
icity can be assigned to the nonmetalated DOTA macrocycle,
which can chelate the body’s cations.[9] The apparent nontoxic-
ity of 5 confirms the in vivo stability of the DOTA–Sm complex.
These results are critically important because they show that
we had succeeded in preparing a stable RuII(arene) complex
that was faithful to known metallodrugs and that could also
be tracked within the body.
Hoping to gain, for the first time, an idea of the uptake, dis-
tribution, and excretion of RuII(arene)-based metallodrugs in
the body, we carried out biodistribution studies of [153Sm]5 in
healthy CD-1 mice. Results were obtained 1 h after administra-
tion and are summarized for the most relevant organs in
Figure 4. The uptake in the tissues was calculated and ex-
Figure 3. HPLC chromatograms of the nonradioactive complex 5 (UV detec-
tion at 254 nm; dotted trace; retention time (tR =12.4 min) and the radioac-
tive analogue [153Sm]5 (g detection; tR =12.5 min). Under the same condi-
tions, the free 153Sm species has an tR value of 2.5 min.
tive analogue 5, which was prepared previously (Figure 3). To
evaluate the stability of the radioactive complex, the radiolab-
eling was done at 708C for 10 min as described above and
then the complex was rapidly cooled and maintained at room
temperature. Under these conditions, the preparation is stable
for at least one day. The complex is also stable in physiological
media (saline, 24 h, at 378C).
Biological experiments
Figure 4. Biodistribution of the 153Sm-labeled Ru(arene) complex 5 at 1 h
after administration to healthy CD-1 mice.
Notably, owing to the metalated DOTA macrocycle, complex 5
is highly water soluble. As discussed above, it is also stable in
biologically relevant media. These properties, in line with the
structural similarity of heterobimetallic theranostic 5 to known
potential metallodrugs, especially RAPTA-type Ru(arene) spe-
cies, encouraged us to proceed with biological evaluation.
The antiproliferative properties of 5 and its nonmetalated
phosphine derivative 1 were assayed by monitoring cell
growth inhibition. Cytotoxic activity was determined on the
cisplatin-resistant human ovarian cancer (A2780cisR) cell line,
after a 72 h exposure to the compounds. From the experimen-
tal data, IC50 values were calculated. Interestingly, the activity
against cisplatin-resistant ovarian carcinoma A2780cisR cells
was significantly higher for RuII(arene) 5 than for phosphine
1 (176 mm versus >300 mm). Notably, for RAPTA-type metallo-
drugs, low IC50 values are not generally observed. Instead, anti-
cancer activity is usually expressed as an overall decrease in
tumor count and size. Thus, the relatively high IC50 value of
176 mm observed here is in line with that expected for this
type of metallodrug.[3e]
pressed as a percentage of the injected radioactivity dose per
gram of tissue (%IDgꢀ1).
The most notable features of the biodistribution study of
[
153Sm]5 are the relatively slow clearance from blood ((2.7ꢁ
0.6)%IDgꢀ1), a fast clearance from soft tissues such as muscle
((0.8ꢁ0.2)%IDgꢀ1), and no relevant uptake in the main
organs, except those related with the excretory paths (liver, in-
testines, and kidneys). The overall rate of radioactivity excre-
tion was relatively low (<40%).
Conclusions
We have successfully synthesized a RuII(arene) complex in-
spired by known therapeutic metal complexes. In contrast to
known variants, the presented complex consists of a pendant
DOTA derivative that can chelate radionuclides, which allows
its location within the body to be determined. The evident
complications in mixing the relatively sensitive nature of RuII
organometallic complexes with the relatively harsh chemistry
and nature of DOTA macrocycles were tackled and overcome.
It is of synthetic note that the heteroditopic ligand 1 can be
easily rutheniated selectively at the phosphorus atom, despite
Finally, we treated several healthy CD-1 mice with theranos-
tic 5 at doses similar to those that would be used in the bio-
distribution study of the radioactive analogue. All of the mice
tested survived this treatment, which showed that 5 does not
have acute toxicity to the organism as a whole. The same
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140.2, 140.4, 166.0, 173.2, 175.3 ppm; HRMS (ESI): m/z calcd for
[C37H47N6O8Pꢀ2H+K]ꢀ: 771.26790; found: 771.26539.
the array of functionalities prone to metal coordination on the
adjacent DOTA cage. Subsequent labeling with radionuclides
occurs without problem. The prepared RuII(arene) complex
shows favorable stability and solubility profiles in biologically
relevant media. Pleasingly, it shows in vitro cytotoxicity in line
with that expected for this type of metallodrug and, just as im-
portantly, it is nontoxic to the organism as a whole. The radio-
labeled complex was tracked in the body to provide informa-
tion about drug uptake, biodistribution, and excretion. This
ability to obtain a big-picture idea of the location of the pre-
sented theranostic sets this work apart from the rest of the
field. As metallotherapeutics develop, a thorough understand-
ing of how biological activity is achieved will be imperative to
designing and improving future metallodrugs. Thus, we be-
lieve strategies such as that shown here will be essential to the
further development of this research field.
Dichloro[(h6-p-cymene)(2,2’,2’’-(10-(2-((2-(4-(diphenylphosphino)-
benzamido)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclodode-
cane-1,4,7-triyl)triacetic acid)-ruthenium(II)] (4): Compound
1
(100 mg, 0.136 mmol) and [RuCl2(p-cymene)]2 (41.6 mg,
0.068 mmol) were dissolved in freshly distilled CH2Cl2 (3.5 mL)
under argon. The solution was stirred at room temperature for
19 h. The solution was concentrated under reduced pressure to
a volume of 1 mL. Freshly distilled pentane (5 mL) was added to
precipitate the product as an orange powder. The powder was fil-
trated under an inert atmosphere and washed with degassed ace-
tone (2ꢄ2 mL) to afford the desired product as an orange-red
solid (0.106 g, 78%). 1H NMR (300.13 MHz, D2O): d=1.09 (d, 6H,
3J=6.9 Hz; CHMe2), 1.87 (s, 3H; Me of p-cymene), 2.82 (brm, 1H;
3
CHMe2 (p-cymene)), 1.95–3.87 (brm, 24H; cyclen), 5.19 (d, 2H, J=
5.9 Hz; CHAr of p-cymene), 5.33 (d, 2H, 3J=5.9 Hz; CHAr of p-
cymene), 7.45–7.96 ppm (m, 14H; CHar), NH signal not observed;
31P{1H} NMR (121.5 MHz, CDCl3): d=24.9 ppm; 13C{1H} NMR
(75 MHz, CDCl3): d=124.3–128.2 (m), 58.2 (s), 52.6 (m), 48.4 (m),
30.3 (s), 22.4 (s), 22.0 (m), 18.4 (s), 17.9 ppm (m); HRMS (ESI): m/z
calcd for [C47H61Cl2N6O8PRuꢀ2H+K]ꢀ: 1077.21951; found:
1077.21750.
Experimental Section
Synthetic procedures
General information: All solvents were dried and distilled under
argon before use. The p-cymene–ruthenium dimer was obtained
by following the procedure of Bennett and Smith.[10] All other re-
agents were commercially available and used as received (DOTA-
derivative A was provided by Chematech). The analyses were per-
formed at the “Plateforme d’Analyses Chimiques et de Synthꢂse
Molꢀculaire de l’Universitꢀ de Bourgogne”. The identity and purity
(ꢂ95%) of the complexes were unambiguously established by
using high-resolution mass spectrometry and multinuclear NMR
spectroscopy. The exact masses of the complexes were obtained
on a Thermo LTQ Orbitrap XL ESI-MS mass spectrometer. 1H
(300.13, 500.13, or 600.23 MHz), 13C (75.5, 125.8, or 150.9 MHz), and
31P (121.5, 202.5, or 242.9 MHz) NMR spectra were recorded on
Bruker 300 Avance III, Bruker 500 Avance III, or Bruker 600 Avance II
spectrometers. Chemical shifts (d) are quoted in parts per million
Dichloro[(h6-p-cymene)(samarium-2,2’,2’’-(10-(2-((2-(4-(diphenyl-
phosphino)benzamido)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraa-
zacyclododecane-1,4,7-triyl)triacetate)-ruthenium(II)]
(5):
Sm(OTf)3 (22 mg, 0.038 mmol) dissolved in dry MeCN (10 mL) was
added to a solution of 4 (35 mg, 0.032 mmol) in dry MeCN (10 mL).
After 36 h, the resulting mixture was filtered and the solid was
washed with dry n-hexane. The desired product was obtained as
a bright orange powder after having been dried under vacuum
(32 mg, 73%). 1H NMR (300.13 MHz, DMSO): d=0.94 (d, 6H, 3J=
6.9 Hz; CHMe2),1.76 (s, 3H; Me of p-cymene), 1.05–3.25 (brm, 25H;
cyclen + CHMe2 (p-cymene)), 5.25 (d, 2H, 3J=5.4 Hz; CHar of p-
3
cymene), 5.32 (d, 2H, J=5.4 Hz; CHAr of p-cymene), 7.05–7.95(m,
14H; CHAr), 8.85 ppm (brs, 2H; HNCH2CH2NH); 31P{1H} NMR
(121.5 MHz, CDCl3): d=24.3 ppm; HRMS (ESI): m/z calcd for
[C48H61Cl2N6O8PRuSmꢀCl]ꢀ: 1154.19830; found: 1154.20347.
1
relative to tetramethylsilane (TMS; H and 13C), by using the residu-
al protonated solvent (1H) or the deuterated solvent (13C) as an in-
ternal standard. Alternatively, 85% H3PO4 (31P) was used as an ex-
ternal standard. The coupling constants are reported in Hertz. All
aromatic positions (ortho, meta, and para) are defined by using
phosphorus as the main group element. Infrared spectra were re-
corded on a Bruker Vector 22 FT-IR spectrophotometer (transmis-
sion mode) with 1% sample mixed with potassium bromide. The
melting points were determined on a BꢃCHI Melting Point B-545
instrument.
Synthesis of radiolabeled complexes
153Sm (T1/2 =46.8 h; bꢀ =0.67 MeV, 34%; 0.71 MeV, 44%; 0.81 MeV,
21%; g=0.103 MeV, 38%) was produced by irradiating isotopically
enriched 152Sm(NO3)3 at the ITN Portuguese Research Reactor. The
nitrate targets were prepared by dissolving 152Sm2O3 with HNO3
and then evaporating to dryness. Irradiation was performed at
1 MW, thermal neutron flux of ~0.8ꢄ1013 neutroncmꢀ2 sꢀ1, and epi-
thermal neutron flux of ~2ꢄ1011 neutroncmꢀ2 sꢀ1. The specific ac-
tivity after 2 h of irradiation and at end of bombardment was
185 MBqmgꢀ1 for 153Sm. After irradiation, the targets were recon-
stituted with water to produce stock solutions for the synthesis of
the complexes. The 153Sm activity was measured in an ionization
chamber (Aloka Curiemeter IGC-3).
2,2’,2’’-(10-(2-((2-(4-(diphenylphosphino)benzamido)ethyl)ami-
no)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triace-
tic acid (1): Compound 3 (0.6 g, 1.34 mmol) was dissolved in fresh-
ly distilled MeCN (30 mL) under argon. Diisopropylethylamine
(DIPEA; 2.3 mL, 13.44 mmol) was then added. The reaction was
stirred at room temperature for 1 h. Compound
2 (0.54 g,
1.34 mmol) was added and the solution was stirred at room tem-
perature overnight. After evaporation of the solvent, the resultant
solid was purified by silica gel column chromatography (eluent:
CHCl3/MeOH/25% NH3·H2O (6:3:1)) to afford the desired product as
The complex [153Sm]5 was prepared by dissolving 4 (4 mg) in
MeOH or DMSO (90 mL) and then adding water (500 mL) and an ad-
equate amount of 153Sm solution (30 mL) to achieve a 1:2 metal/
ligand molar ratio. The final pH value was ~5.5 and the final ligand
concentration was 6.3 mm. The radiolabeling efficiency and reac-
tion kinetics of the radiocomplexes were determined by RP-HPLC
and by ITLC-SG analysis by using silica gel (ITLC-SG) strips (Poly-
gram, Macherey–Nagel) with MeOH/H2O/conc. aq. NH3 (2:4:0.2)
and MeOH/6m HCl (95:5) as the eluents. The radioactive distribu-
1
a white solid (0.56 g, 57%). H NMR (300 MHz, MeOD): d=2.0–3.56
(brm, 24H; cyclen), 7.29–7.84 ppm (m, 14H; CHar), NH signal not
observed; 31P{1H} NMR (121.5 MHz, CDCl3): d=ꢀ5.7 ppm;
13C{1H} NMR (75.5 MHz, D2O): d=48.3, 52.5, 56.4, 59.6, 127.6, 127.7,
128.8, 128.9, 129.2, 132.6, 132.9, 133.2, 133.5, 134.7, 136.0, 136.1,
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tion on the ITLC-SG strips was detected by using a Berthold LB 505
g detector coupled to a radiochromatogram scanner.
Acknowledgements
Support was provided by the Regional Council of Burgundy
(France) (PARI & 3MIM programs), the French Ministry of Higher
Education and Research (MESR), and the French National Centre
for Scientific Research (CNRS). Ms. M.-J. Penouilh (PACSMUB) is
gratefully acknowledged for HRMS analysis. F.M. thanks the Por-
tuguese Foundation for Science and Technology (FCT) for her FCT
Investigator grant.
HPLC analyses of the inactive and radioactive complexes were ach-
ieved on a reverse-phase EC-Nucleosil C18 column (250ꢄ4 mm,
10 mm) eluted at a flow rate of 1.0 mLminꢀ1 with 0.1% trifluoroace-
tic acid (TFA) aqueous solution (A) and 0.1% TFA in MeCN (B) as
the eluents and with the following linear gradient: 0–3 min,
100%A/0%B; 3–18 min, 100%A/0%B!0%A/100%B; 18–20 min,
0%A/100%B.
Keywords: heterometallic complexes
·
macrocycles
·
phosphines · radiopharmaceuticals · theranostics
Cytotoxicity assays
Cell culture: The human ovarian cancer cell line A2870cisR (resistant
to cisplatin; ECACC, UK) was grown in RPMI 1640 culture medium
(Invitrogen) supplemented with 10% fetal bovine serum (FBS) and
1% penicillin/streptomycin at 378C in a humidified atmosphere of
95% of air and 5% CO2 (Heraeus, Germany).
[1] a) B. Rosenberg, L. Vancamp, T. Krigas, Nature 1965, 205, 698; b) B.
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Received: November 22, 2013
Published online on January 21, 2014
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ChemMedChem 2014, 9, 1567 – 1573 1573