Synthesis of the first radiolabeled 188Re N-heterocyclic carbene complex and initial studies on its potential use in radiopharmaceutical applications

Article abstract of DOI:10.1002/jlcr.3203

A novel approach towards the synthesis of radiolabeled organometallic rhenium complexes is presented. We successfully synthesized and analyzed the first ¹⁸⁸Re-labeled N-heterocyclic biscarbene complex, trans-dioxobis(1,1′-methylene-bis(3,3′-diisopropylimidazolium-2- ylidene))¹⁸⁸rhenium(V) hexafluorophosphate (¹⁸⁸Re-4) via transmetalation using an air-stable and moisture-stable silver(I) biscarbene complex. In order to assess the viability of this complex as a potential lead structure for in vivo applications, the stability of the ¹⁸⁸Re-NHC complex was tested in physiologically relevant media. Ultimately, our studies illustrate that the complex we synthesized dissociates rapidly and is therefore unsuitable for use in radiopharmaceuticals. However, it is clear that the transmetalation approach we have developed is a rapid, robust, and mild method for the synthesis of new ¹⁸⁸Re-labeled carbene complexes. Copyright

Full text of DOI:10.1002/jlcr.3203

Research Article
Received 25 February 2014,
Revised 27 March 2014,
Accepted 1 April 2014
Published online 29 May 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3203
1
88
Synthesis of the first radiolabeled Re
N-heterocyclic carbene complex and initial
studies on its potential use in
radiopharmaceutical applications
a
b
c
a
Thomas Wagner, Brian M. Zeglis, Sam Groveman, Claudia Hille,
a
c
a
Alexander Pöthig, Lynn C. Francesconi, Wolfgang A. Herrmann,
a
b,d
Fritz E. Kühn, * and Thomas Reiner **
A novel approach towards the synthesis of radiolabeled organometallic rhenium complexes is presented. We successfully
1
88
synthesized and analyzed the first 1 Re-labeled N-heterocyclic biscarbene complex, trans-dioxobis(1,1′-methylene-bis(3,3′-
88
188
diisopropylimidazolium-2-ylidene)) rhenium(V) hexafluorophosphate ( Re-4) via transmetalation using an air-stable
and moisture-stable silver(I) biscarbene complex. In order to assess the viability of this complex as a potential lead structure
1
88
for in vivo applications, the stability of the
Re-NHC complex was tested in physiologically relevant media. Ultimately, our
studies illustrate that the complex we synthesized dissociates rapidly and is therefore unsuitable for use in
radiopharmaceuticals. However, it is clear that the transmetalation approach we have developed is a rapid, robust, and mild
1
88
method for the synthesis of new
Re-labeled carbene complexes.
1
88
Keywords: Re carbene complex; radiopharmaceutical application; N-heterocyclic carbene; transmetalation
1
86/188
Introduction
Despite these difficulties,
Re remain promising isotopes
for nuclear medicine applications. Due to their emission of high-
energy β particles that can penetrate several millimeters into
solid tumors ( Re: 1.069 MeV, 92.5%;
the two radiometals have significant potential as
Moreover, based on the energies of the
γ-photons emitted from each isotope—137 and 155 keV for
For almost half a century, radioactive isotopes of technetium and
rhenium have received increasing attention for use in both
diagnostic and therapeutic radiopharmaceuticals. Technetium
has one medically useful isotope,
in contrast, has two isotopes used in nuclear medicine,
À
186
188
Re: 2.12 MeV, 100%),
1
,2
99m
Tc (t1/2 = 6.0 h). Rhenium,
Re
Re (t1/2 = 17.0 h), that have potential in
6–11
radiotherapeutics.
186
188
(t
1/2 = 3.72 days), and
186
188
Re and
Re, respectively—both radioisotopes could be
both diagnostic and therapeutic applications. The two metals
are congeners in Group VIIB, and as such, there are a number
of important similarities between their coordination chemistries.
Indeed, this similarity in fundamental chemistry has led many to
envision the use of the two radiometals as a ‘matched pair’ for
identified during radiotherapy by single photon emission
computed tomography imaging.
186
188
Several Re-based or Re-based radiopharmaceuticals have
been tested in preclinical studies, and some of them have been
9
9m
186/188
imaging ( Tc) and therapy (
the same ligand scaffold.
Re) using vectors bearing
a
Chair of Inorganic Chemistry/Molecular Catalysis, Department of Chemistry and
Catalysis Research Center, Technische Universität München, Garching b.
München, Germany
However, any list of radiopharmaceuticals used in the clinic
quickly reveals that the two radiometals are not utilized with
9
9m
b
Radiochemistry and Imaging Sciences Service, Department of Radiology,
similar frequency:
Tc has been widely used in a number of
Memorial Sloan Kettering Cancer Center, New York, NY, USA
diagnostic single photon emission computed tomography
1
86
188
applications, whereas Re and Re have not been employed
c
Hunter College, New York, NY, USA
1
,3
with equal success. This disparity is in part due to a small but
fundamental difference between the chemistry of the two
metals. Upon coordination, rhenium is both harder to reduce
d
Center for Molecular Imaging and Nanotechnology, Memorial Sloan Kettering
Cancer Center, New York, NY, USA
and easier to oxidize under biologically relevant conditions than *Correspondence to: Fritz E. Kühn, Chair of Inorganic Chemistry/Molecular
technetium. This susceptibility to redox chemistry reduces the Catalysis, Department of Chemistry and Catalysis Research Center, Lichtenbergstr.
4
, Technische Universität München, Garching b. München, Germany.
E-mail: fritz.kuehn@ch.tum.de
*Correspondence to: Thomas Reiner, PhD, Department of Radiology, Memorial
stability of rhenium chelate complexes, which—in concert with
186
the low specific activity of Re and the successful clinical use
*
9
0
177
4
of Y and
Lu isotopes —has consequently hampered the
Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065.
E-mail: reinert@mskcc.org
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5
development of
Re-based radiopharmaceuticals.
J. Label Compd. Radiopharm 2014, 57 441–447
Copyright © 2014 John Wiley & Sons, Ltd.

T. Wagner et al.
8
–11
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successfully translated to the clinic.
Specifically,
Re- Experimental
hydroxylethylene diphosphonate complexes are currently being
1
1,12
186/
Unless otherwise indicated, all solvents and reagents were obtained from
Sigma-Aldrich (St. Louis, MO, USA) or VWR International GmbH
used as palliative therapeutics for bone metastases,
and
Re-complexes with thiolate-functionalities and amine/amide-
functionalities can be used for the treatment of hepatocellular
188
(Darmstadt, Germany) and used without further purification. 18.2 MΩ/cm
water was obtained from a PURELAB ultra device (ELGA LLC., Woodridge, Il,
USA). N-Isopropylimidazole, 3,3′-diisopropyl-1,1′-methylenebisimidazolium
10,13–16
carcinoma (Figure 1(A–C)).
3
Tetradentate N S type complexes
(
Figure 1(D)) have been especially popular as chelate scaffolds for
bis(hexafluorophosphate),
In addition, various organometallic bis-(imidazole-2-ylidene)silver(I)] bis(hexafluorophosphate) (3),
rhenium(I)-tricarbonyl complexes have also proven suitable trichloro-oxo-bis(triphenylphosphine)rhenium(V), and Dioxobis(1,1′-
bis-[3,3′-diisopropyl-1,1′-methylene-
17,18
radiolabeled antibodies.
19,20
chelation architectures (Figure 1(E)).
While most targeted methylene-bis(3,3′-diisopropylimidazolium-2-ylidene))rhenium(V)
hexafluorophosphate (4) were synthesized similar to the previously
Re are based on chelate complexes, the
1
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approaches with
isotopes have also been applied in a colloidal form,
2
9,37–40
188
21,22
published procedures.
Samples of Re were obtained from a 10-
188
similar to
1
88
99m
23–25
month old, 10.8GBq W/ Re-generator (Isotope Technologies Garching
GmbH, Garching, Germany) by elution using 10mL of 0.9% sterile
Tc sulfur colloids.
While these successes no doubt represent steps in the right
direction, more stable and easier-to-use rhenium chelators must
be developed in order for the radiometal to fulfill its promise as a
diagnostic and therapeutic radioisotope. Ultimately, the goal is
sodium chloride solution (Hospira Inc., Lake Forest, Il, USA) at a flow rate
of 1mL/min. The eluate is concentrated by passing over a Waters Sep-Pak
Accell Plus QMA column (Milford, MA, USA) and loading of the activity to
a 2.5mL Dionex OnGuard II Ag (Thermo Scientific, Waltham, MA, USA)
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simple: the creation of a
Re chelator that allows rapid, mild, column. Subsequent elution with 2 mL of 0.9% sterile sodium chloride
and cofactor-free attachment of the radiometal to complex solution at a flow rate of 1mL/min yielded the final Na ReO solution
4
188
biomolecular vectors.
(695 Ci/mg).
Electrospray ionization mass spectra were recorded with a Waters
Acquity UPLC (Milford, MA, USA) with electrospray ionization SQ detector.
High-resolution mass spectra (HRMS) were recorded with a Waters LCT
Premier system (ESI). HPLC experiments were performed on a Shimadzu
Prominence UFLC system equipped with (i) a flow cell Posi-Ram Model 4
radiodetector from LabLogic (Brandon, FL), Inca DGU-20A degasser, two
LC-20AB pumps, a SPD-M20A photodiode array detector (190–600 nm
bandwidth) and a SPD-M20A autosampler or (ii) a Flow-Ram Radio HPLC
With this aim in mind, we recently became interested in
188
investigating new chelation architectures for
decided to focus on the use of organometallic
bonds, specifically those seen in N-heterocyclic carbene (NHC)
Re. We have
188
Re-carbon
26–29
complexes of the metal.
the synthesis of transition metal complexes in the late 1960s,
this class of ligands has attracted a significant amount of
Since the introduction of NHCs for
30
31
attention in the field of organometallic catalysis. Indeed, the detector from LabLogic (Brandon, FL), Inca DGU-20A degasser, two
study of organometallic NHC complexes has uncovered a LC-20AB pumps and a SPD-M20A photodiode array detector. In either case,
32
number of exceptional catalysts, a success which owes quite a a reversed phase Phenomenex® Luna 5μm C18, 100Å, 250 × 4.6mm
analytical column was used. Radioactivity measurements were performed
with a Capintec CRC1243 Dose Calibrator (Capintec, Ramsay, NJ, USA).
bit to the discovery of several synthetic routes towards these
33–35
ligand systems which increased their diversity and availability.
To the best of our knowledge, NHC ligand scaffolds have not
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yet been applied to the coordination of
have recently been used for the complexation of Tc.
Re, though they N-isopropylimidazole (1)
99
36
N-isopropylimidazole was synthesized with slight modification according
Therefore, in the work at hand, we aim to present a synthetic
37,38
to procedures reported earlier.
Briefly, a solution of isopropylamine
approach towards the first NHC complex of generator-produced
(
14.8 g, 250 mmol, 1.00 eq) in methanol (30 mL) was added within
188
Re and to assess its viability as a radiotracer for clinical use.
9
1
0 min to a cooled (0 °C) slurry of paraformaldehyde (7.89 g, 263 mmol,
.05 eq) in methanol (30 mL). Subsequent addition of an aqueous
glyoxal-solution (40%, 36.3 g, 250 mmol, 1.00 eq) containing ammonium
carbonate (12.0 g, 125 mmol, 0.50 eq) and methanol (60 mL) led to an
orange solution, which was warmed to ambient temperature and stirred
for 16 h. The solution was concentrated to yield a reddish-brown, viscous
À2
liquid. The product was obtained by vacuum distillation (2.2 × 10 mbar,
1
5
(
5 °C) as a clear, almost colorless liquid (2.60 g, 23.6 mmol, 19%). H NMR
2
400 MHz, CDCl ): δ (ppm) = 7.55 (s, 1H), 7.04 (t, JHH = 1.1 Hz, 1H), 6.95
d, JHH = 1.3Hz, 1H), 4.34 (hept, JHH = 6.7 Hz, 1H), 1.48 (d, JHH = 6.7Hz, 6H).
3
2
3
3
(
1
3
C (101 MHz, CDCl
3
): δ (ppm) = 135.1, 128.9, 116.6, 49.2, 23.7, 22.7. ESI-HRMS
+
(
m/z): calculated for C H N ([M + H] ): 111.0922, found: 111.0917.
6 11 2
1,1′-Methylene-bis(3,3′-diisopropylimidazolium)
dihexafluorophosphate (2)
The title compound was synthesized analog to procedures reported
3
9
earlier.
Briefly, N-isopropylimidazol (2.56 g, 23.3 mmol, 0.50 eq),
dibromomethane (2.01 g, 11.6 mmol, 1.00 eq), and THF (10 mL) were
placed in a pressure tube and heated to 130 °C with stirring for 72 h.
After cooling to room temperature, the white solids were filtered off
and washed with THF (3 × 30 mL) and diethyl ether (3 × 10 mL). The
solids were redissolved in water, and an excess of ammonium
hexafluorophosphate was added as a saturated, aqueous solution with
stirring. The resulting white slurry was stirred for another 1 h at an
ambient temperature before the off-white raw product was filtered off
and washed with water (3 × 30 mL). Dissolution in a small amount of
1
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Figure 1. General design of
Re tracers that already have been used in
clinical applications: (A) N2S2-type amine/amidodithiolato ligand on an
oxorhenium(V) core. (B) NS3-type combination of amidodithiolate and thiolato
ligands on an oxorhenium(V) core. (C) DMSA or DMSA derivatives as stabilizing
ligand for oxorhenium(V) cores. (D) Oxorhenium(V) core with an N3S-type or
MAG3 ligand, used for coupling to biomacromolecules. (E) Rhenium(I)tricarbonyl
core structure with three-coordinate bifunctional ligands, mostly used for coupling
biomacromolecules. (F) Schematic drawing of a rhenium/sulfur colloid used, for
1
5
1
4
1
3
1
7
2
0
2
1
example, in radio-synovectomies.
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 441–447

T. Wagner et al.
À6
À7
acetonitrile and precipitation with diethyl ether yielded a white solid dichloromethane (422 ± 28 μCi, 2.21 × 10 ±1.48×10 μmol, 82.7 ± 4.3%,
which was filtered, washed with diethyl ether (3 × 10 mL), and dried 229 Ci/mg, n= 10).
under reduced pressure to give 6.10 g (11.6 mmol, 85%) of 2 as white
1
powder. H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.66 (br s, 2H), 8.10
Dioxobis(1,1′-methylene-bis(3,3′-diisopropylimidazolium-2-
3
4
3
4
(
6
dd, JHH =1.9Hz, JHH = 1.9 Hz, 2H), 8.04 (dd, JHH =1.9Hz, JHH = 1.9 Hz, 2H),
188
+ 188
ylidene))rhenium(V), [ Re(O)
2
L
2
] ( Re-4)
3
3
.67 (s, 2H), 4.71 (hept, JHH = 6.7 Hz, 2H), 1.51 (d, JHH =6.7Hz, 12H). ESI-HRMS
+
188
3 3 2
An aliquot of 0.1 mL of freshly prepared ReOCl (PPh ) (196 ± 14.3 μCi,
(
m/z): calculated for C13
Elemental analysis: calculated for C13
N 11.32%; found: C 31.71%; H 4.16%; N 11.24%.
H
22
F
6
N
4
P ([MÀ PF
6
] ): 379.1486, found: 379.1486.
À6
À8
H
22
F
12
N
4
P
2
: C 32.16%; H 4.16%; 1.03 × 10 ± 7.50 × 10 μmol, n = 6) was added to a freshly prepared
suspension of ReOCl (PPh ) (1.25 mg, 1.50 μmol) and the silver carbene
3
3 2
3
(1.45 mg, 1.5 μmol) in acetonitrile (0.5 mL). The mixture was vigorously
Di(1,1′-methylene-bis(3,3′-diisopropylimidazolium-2-ylidene)) shaken for 2 h at ambient temperature, followed by filtration of
precipitates. The crude mixture (146 ± 13.8 μCi, 75 ± 1.7%, RCP
disilver(I) dihexafluorophosphate (3)
9
2 ± 4.5%, specific activity 146 ± 14.0 μCi/mg, n = 6) was purified via
The title compound was synthesized similar to procedures described HPLC, yielding the analytically pure title compound, containing
2
9
188
+
earlier. Silver(I)oxide (6.72 g, 29.0 mmol, 2.50 eq) was added to a
2 2
approximately 40 μCi/mL [ Re(O) L ] .
solution of bisimidazolium salt (6.10 g, 11.6 mmol, 1.00 eq) in
2
acetonitrile (50 mL). The reaction mixture was stirred for 4 h at an
ambient temperature under exclusion of light. After removal of residual
Complex stability determinations
1
88
Ag
concentrated to 10 mL. Addition of diethyl ether led to the precipitation
of an off-white solid, which was filtered off and washed with diethyl ether pH 7), and in fetal bovine serum (FBS) for incubation times of up to 4 h.
Samples were prepared for each time point and each substrate by adding
2
O by filtration over a pad of Celite®, the clear solution was Stability testing of Re-4 was carried out in water at different pH values
(
pH 5, pH 6, pH 7, and pH 8), in PBS at different pH values (pH 5, pH 6, and
(
3 × 30 mL). The raw product was redisolved in acetonitrile (10 mL) and
188
+
5
0 μL of a freshly prepared solution of [ Re(O)
of the appropriate medium (H O, PBS, or FBS). Samples were incubated at
7 °C whilst agitated for 1 h, 2 h, 3 h, and 4h. After incubation, 200 μL of
2 2
L ] (~40 μCi/mL) to 500 μL
again precipitated by addition of diethyl ether. The now white solid
was collected by filtration, washed with diethyl ether (3 × 30 mL), and
2
3
dried under reduced pressure to yield 4.12 g (4.24 mmol, 73%) of 3 as
1
an off-white, flaky powder. H NMR (400 MHz, DMSO-d6): δ (ppm) = 7.88 each sample solution were subjected to analysis using an HPLC coupled
3
188
(
br s, 4H), 7.73 (d, JHH = 1.9Hz, 4H), 6.83 (br s, 2H), 6.40 (s, 2H), 4.68 (hept, with a Posi-Ram radiodetector for the determination of Re-4 stability.
3 13
3
J
HH = 6.7Hz, 4H), 1.43 (d, JHH = 6.7Hz, 24H). C NMR (101 MHz, DMSO-d6):
δ (ppm) = 122.1, 119.8, 63.6, 54.2, 23.1. ESI-mass spectrometry (MS) (+, m/z): Variation of carrier concentration
+
calculated for C26
H
40Ag
2
F
6
N
8
P ([M À PF
6
] ): 825.11, found: 825.2; ESI-MS
À
The influence of cold carrier on the radiochemical yield of the
transmetalation reaction was tested adding different amounts of cold
(À, m/z): calculated for C26
H
40Ag
2
F
18
N
8
P
3
([M + PF
6
] ): 1115.04, found:
+
1115.0. ESI-HRMS (m/z): calculated for C26
H40Ag
2 6
F N
8
P ([M-PF
6
] ): 823.1120,
ReOCl
following the synthetic protocol of [ Re(O)
amounts of cold rhenium. Carrier concentrations of 0.0 μmol (0.00 mg),
.5 μmol (0.42 mg), 1.0 μmol (0.83mg), 1.5μmol (1.25 mg), and 2.0μmol
(1.67mg) of cold ReOCl (PPh were added. Simultaneously, the amounts
of silver carbene 3 were adjusted to maintain molar ratios. In case of the
carrier-free reaction, 0.5μmol (0.48 mg) of 3 was used. Yields were
determined by HPLC analysis of the reaction mixtures, collection of the
fractions, and determination of their activities.
3 3 2
(PPh ) to the reaction solution. The reactions were performed
found: 823.1142. Elemental analysis: calculated for C26
C 29.78%; H 4.23%; N 10.69%; found: C 29.61%H 4.39%; N 10.31%.
2 12 8 2
H40Ag F N P :
188
+ 188
2
L
2
]
Re-4, with varying
0
Dioxobis(1,1′-methylene-bis(3,3′-diisopropylimidazolium-2-
+
3
3 2
)
ylidene))rhenium(V), [Re(O) L ] (4)
2
2
The title compound was synthesized with slight modification according
29
to
a
previously published procedure
:
ReOCl
3
(PPh
3
)
2
(21.4 mg,
2
1
5.7 μmol, 1.00 eq) and the silver carbene 3 (25.1 mg, 25.9 μmol,
.00 eq.) were placed in a round bottomed flask. After addition of
acetonitrile (5 mL), the reaction was stirred at room temperature for 2 h Results and discussion
before AgPF (6.5 mg, 25.8 μmol, 1.00 eq) was added and the brownish
suspension was stirred for another 30 min at room temperature. The A particularly promising family of structures in the pursuit of
6
188
crude mixture was filtered to remove AgCl, diluted with toluene (5 mL), stable Re-NHC bioconjugates is found in trans-dioxorhenium NHC
concentrated under reduced pressure to a total volume of approximately complexes. Both their pseudo-octahedral coordination geometry—
4
mL, and again diluted with toluene (5 mL). Upon final concentration to
which provides steric shielding of the metal center—and the
closed-shell electronic structure of the dioxorhenium(V) metal center
a total volume of 4 mL, the title compound crashed out of the reaction
mixture and was subsequently isolated by filtration. Washing with small
portions of diethyl ether and drying under reduced pressure gave 4
as brownish solid (10.1 mg, 10.2 μmol, 40%). H NMR (400 MHz, CD
account for the high stability of these complexes towards moisture,
26–28,41
1
thermal stress, nucleophilic attack, and oxidation.
In addition,
3
CN):
HH = 2.0 Hz, 4H), 6.85
HH = 13.6 Hz, 2H), 4.42 (br s, 4H), 1.54
3
3
the accessibility of these complexes via transmetalation from stable
δ (ppm) = 7.73 (d,
JHH = 2.0 Hz, 4H), 7.50 (d, J
29
2
2
silver(I) carbene complexes abrogates the need for the in situ
(d, 3JHH = 13.6 Hz, 2H), 6.38 (d,
(d,
J
3
13
generation of free carbenes, which is often associated with the
JHH = 6.6 Hz, 12H), 1.01 (d, JHH = 6.4 Hz, 12H). C NMR (100 MHz,
33,34
CD
calculated for C26
CN): δ (ppm) = 168.6, 125.0, 120.0, 65.7, 54.2, 25.2. ESI-HRMS (m/z): addition of cofactors and harsh reaction conditions.
3
+
H
41
F
12
N
8
O
2
P
2
Re ([M + H] ): 684.2910, found: 684.2883.
The synthesis of the trans-dioxorhenium biscarbene complex
4
—reported by Hor and coworkers in 2013—served as the
1
88
188
Trichloro-oxo-bis(triphenylphosphine)rhenium(V), ReOCl3
starting point for the development of a route towards a
Re-
1
88
29
(
PPh ) ( Re-5)
labeled biscarbene complex. Due to the relative inertness of
trans-dioxorhenium carbene complexes towards air and
moisture, the cold rhenium carbene complex 4 was synthesized
3
2
The compound was synthesized according to a previously published
4
0
188
À
4
method. An aliquot of 0.1 mL of the
2
ReO
.68 × 10 ± 1.44 × 10 μmol, 695 Ci/mg, n = 10) was added to a mixture
of PPh (3.33 mg, 12.7 μmol) in HCl (0.3 mL, 37%) to yield a 9 M HCl
solution. After addition of DCM (0.3 mL), the biphasic mixture was
agitated for 5 min at an ambient temperature. Removal of the aqueous temperature in a mixture of acetonitrile and water, and no
eluate (511 ± 27.5 μCi,
26,27
À6
À7
under aerobic atmosphere.
It was generated from the
commercially available complex ReOCl (PPh ) in the presence
3
3 2
3
of the silver carbene 3 (Figure 2(A)). The complex is stable at room
188
phase yielded the product
ReOCl
3
(PPh
3
)
2
as a solution in decomposition was observed up to 24 hours. Complex 4 was
J. Label Compd. Radiopharm 2014, 57 441–447
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

T. Wagner et al.
Figure 2. Synthesis and identification of cold dioxobis(1,1′-methylene-bis(3,3′-diisopropylimidazolium-2-ylidene))rhenium(V) hexafluorophosphate, 4. (A) Reaction
scheme for the transmetalation reaction to yield compound 4. (B) Mass spectrometry of compound 4. (C) High-resolution mass spectrometry, displaying the exact mass
and the isotopic pattern of 4 and its protonated, dicationic form. (D) UV-spectrum of compound 4 with a strong absorption band at 310 nm.
1
13
188
identified via H and
C-NMR as well as via MS and dichloromethane to give the intermediate product
ReOCl
Re-5, in 83 ± 4% yield with a reaction time of only
Figure 2(B,C)). The absorbance of the complex at 310 nm was 5 min. In the second step, a solution of silver(I) carbene 3 in
attributed to a metal-to-ligand charge transfer band which acetonitrile was added to the dichloromethane extract containing
3
1
88
HRMS, confirming the elemental composition described earlier (PPh ) ,
3 2
40
2
9
(
1
88
À6
À7
has been previously identified for other octahedral trans- 378 ± 23.8 μCi Re-5 (1.99 × 10 ±1.25 × 10 μmol, 229 Ci/mg).
dioxorhenium compounds.
42
The reaction mixture was then agitated for 2 h at room
29
Importantly, in stark contrast to the synthesis of the cold temperature. Initial experiments showed that this procedure
1
88
188
complex 4, the Re-labeled counterpart has to be synthesized yields
Re-4 in high specific activity (278 Ci/mg). After HPLC
), the Re species produced purification of Re-4 using acetonitrile and water as eluent, the
188
À
188
188
starting from perrhenate ( ReO
4
1
88
188
by
decrease preparation time, we sought to synthesize the
carbene species in a procedure without intermediate work-up
W/ Re-generators. In order to increase yields and title compound was obtained in 6.7± 7.8% radiochemical yield
188
Re and >98% radiochemical purity.
Based on these somewhat meager radiochemical yields, it was
188
188
188
(
Figure 3(A)). First, an aliquot of the W/ Re-generator eluent was reasoned that the tracer amounts of
ReOCl (PPh ) in
3 3 2
À6
À7
obtained containing 511 ± 27.5 μCi (2.68 × 10 ±1.44×10 μmol, combination with impurities or188 side reactions could
1
88
À
4
6
95 Ci/mg) of
ReO in sterile 0.9% saline solution. This ultimately reduce the amount of
ReOCl (PPh ) available
3 3 2
eluent was mixed under ambient conditions with an excess of for transmetalation and consequently reduce the final yields.
triphenylphosphine in concentrated hydrochloric acid and This hypothesis was further supported by experiments in which
1
88
188
Figure 3. Synthesis of trans-dioxobis(1,1′-methylene-bis(3,3′-diisopropylimidazolium-2-ylidene)) rhenium(V) hexafluorophosphate,
Re-4. (A) Reaction scheme of the
188
188
synthesis of compound
Re-4. (B) UV and (C) radio HPLC signal of a purified sample of compound
Re-4.
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T. Wagner et al.
3 3 2
cold carrier ReOCl (PPh ) was added to the reaction mixture. In
these cases, the yield of the transmetalation reaction forming
1
88
Re-4 was significantly increased, culminating in 62 ± 5%
radiochemical yield with 1.0 μmol of added cold carrier (Figure 4(A)).
After HPLC purification of the crude reaction mixtures, we
1
88
obtained analytically pure solutions of
activities up to 386.4 ± 1.5 μCi/mg and a radiochemical purity
3 2
98% (Figure 3(B,C)). Interestingly, amounts of cold ReOCl (PPh )
Re-4 with specific
>
3
carrier greater than 1.0 μmol did not lead to a further increase in
yield but rather simply resulted in a reduction of specific activity
(208.5 ± 2.5 μCi/mg and 191.5 ± 5.0 μCi/mg for 1.5 μmol and
2.0 μmol of cold carrier, respectively; Figure 4(B)).
Complex 4 is stable to air and moisture, one of the
fundamental prerequisites for a successful chelation scaffold.
However, in order to explore the utility of its radiolabeled
1
88
counterpart,
levels between 5.0 and 8.0 at 37 °C (Figure 4(C)). The HPLC
analysis of
Re-4 was incubated in water at different pH
1
88
Re-4 in aqueous solutions over a period of 4 h
revealed that approximately 25% of the rhenium complex
decomposes within the first hour regardless of the pH value.
After that point, decomposition is significantly slowed for the
samples at pH 5.0, while for the samples at pH 6.0 and above, less
1
88
than 50% of the initial amount of
four hours at 37 °C (Figure 4(C)). While decreasing amounts of
Re-4 remained intact after
188
188
Figure 5. Stability of
Re-4. Radio-HPLC of
Re-4 in H2O (A), PBS (B), and FBS
188
188
Re-4 were observed in these samples (t = 12.2 min, Figure 5(A)),
R
(C) after incubation at 37 °C for 1, 2, 3, and 4 h, respectively. Green arrow:
Re-4;
1
88
-
the concomitant increase in any other radioactive peaks was not Red arrow: ReO4 .
witnessed in the HPLC chromatogram. This strongly suggests that
1
88
À
reoxidation to free
ReO is not the major decomposition
4
1
88
À
pathway, as free ReO would appear in the chromatogram at In PBS solutions at three different pH values—pH 5.0, 6.0, and 7.0—
= 7.5min. Rather, it was observed that increasing amounts of the decomposition of Re-4 was found to be rapid, with almost
activity were trapped on the HPLC column after decomposition none of the radiolabeled complex remaining intact after 2 hours
of
Re-4. However, when compared to the recently published, at 37 °C (Figure 5(B)). This finding may be attributed to the fact that
similar Tc complex, the difference in stability against aqueous the ionic strengths of the PBS solutions are significantly higher than
4
1
88
t
R
1
88
9
9
36
4
6
media is considerable. Apart from the different metal center, those of non-buffered water of the same pH value (overall, 10 –10 -
1
88
the observed lower stability of
bulkiness of the isopropyl substituents and the terminal oxo- anions, especially the chloride ions, are able to compete with and
ligands, facilitating the de-coordination of the ligand.
displace the carbenes at the metal center. In this case, the resultant
In the next step, the stability of Re-4 in solutions of phosphate free carbenes would immediately form imidazolium salts, which
buffered saline (PBS; 12 mM PO
Re-4 may be attributed to the fold increase in ion concentration). It can be assumed that the
188
3
À
À
26
, 139.7 mM Cl ) was investigated. would be unable to bind to the rhenium once again.
4
1
88
188
Figure 4. Yields, specific activities, and stability of
Re-4 in aqueous solution. (A) Effect of added cold carrier ReOCl
3
(PPh
)
3 2
on the yield of compound
Re-4.
188
188
(
B) Specific activity of1 Re-4 at different amounts of added cold carrier. (C) Stability of
Re-4 in H2O at different pH values over time. Peak areas are normalized to
88
the initial amount of
Re-4.
J. Label Compd. Radiopharm 2014, 57 441–447
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

T. Wagner et al.
1
88
Finally, the stability of
Re-4 was investigated in fetale Conflict of Interest
bovine serum, a medium that provides a reasonable facsimile
of the in vivo environment. Perhaps not surprisingly, the
The authors did not report any conflict of interest.
1
88
stability of
Re-4 in FBS was similar to that in PBS. Here,
almost complete decomposition of the rhenium carbene References
complex was observed within the first hour of incubation
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[
[
1
88
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1
88
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88
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Conclusion
The principal aims of this study were to develop
a
methodology for the synthesis of radioactive, organometallic
1
88
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1
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188
chelation of both
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