Expedient multi-step synthesis of organometallic complexes of Tc and Re in high effective specific activity. A new platform for the production of molecular imaging and therapy agents

Article abstract of DOI:10.1021/ic800775w

For over thirty years, instant labeling kits which involve no purification steps have been the only method used to prepare ^99mTc radiopharmaceuticals for clinical studies. To address the limitations imposed by instant kits, which is hindering the development of molecularly targeted Tc- and Re-based imaging and therapy agents, a new strategy for the rapid multistep synthesis and purification of organometallic technetium-based molecular probes and corresponding rhenium-based therapeutic analogues was developed. Beginning with MO₄^- (M = ^99mTc, ^186/188Re), the carbonyl precursor [M(CO)₃(H₂O)₃] ⁺ was synthesized in 3 min in quantitative yield in a microwave reactor. A dipicolyl ligand was added and the chelate complex was formed in high yield in 2 min using microwave heating at 150°C. This was followed by a new purification strategy to remove unlabeled ligand which entailed using a copper resin/C18 solid phase extraction protocol giving the desired product in greater than 78% decay corrected yield (dcy). Conversion to the corresponding succinimidyl active ester was achieved following a 5 min microwave irradiation at 120°C and C18 solid phase extraction purification in 60% dcy. A series of amides were prepared subsequently by microwave heating at 120°C for 5 min producing the desired targets in greater than 86% dcy. The reported method represents a move away from traditional instant kits toward more versatile platform synthesis and purification technologies that are better suited for producing modern molecular imaging and therapy agents.

Full text of DOI:10.1021/ic800775w

Inorg. Chem. 2008, 47, 8213-8221
Expedient Multi-Step Synthesis of Organometallic Complexes of Tc and
Re in High Effective Specific Activity. A New Platform for the
Production of Molecular Imaging and Therapy Agents
Patrick W. Causey,† Travis R. Besanger,‡ Paul Schaffer,† and John F. Valliant*,‡,§
McMaster Nuclear Reactor, Department of Chemistry, and McMaster Institute of Applied
Radiation Sciences (McIARS), McMaster UniVersity, Hamilton, Canada, L8S 4M1
Received April 29, 2008
For over thirty years, instant labeling kits which involve no purification steps have been the only method used to
prepare 99mTc radiopharmaceuticals for clinical studies. To address the limitations imposed by instant kits, which
is hindering the development of molecularly targeted Tc- and Re-based imaging and therapy agents, a new strategy
for the rapid multistep synthesis and purification of organometallic technetium-based molecular probes and
corresponding rhenium-based therapeutic analogues was developed. Beginning with MO4- (M ) 99mTc, 186/188Re),
the carbonyl precursor [M(CO)3(H2O)3]+ was synthesized in 3 min in quantitative yield in a microwave reactor. A
dipicolyl ligand was added and the chelate complex was formed in high yield in 2 min using microwave heating at
150 °C. This was followed by a new purification strategy to remove unlabeled ligand which entailed using a copper
resin/C18 solid phase extraction protocol giving the desired product in greater than 78% decay corrected yield
(dcy). Conversion to the corresponding succinimidyl active ester was achieved following a 5 min microwave irradiation
at 120 °C and C18 solid phase extraction purification in 60% dcy. A series of amides were prepared subsequently
by microwave heating at 120 °C for 5 min producing the desired targets in greater than 86% dcy. The reported
method represents a move away from traditional instant kits toward more versatile platform synthesis and purification
technologies that are better suited for producing modern molecular imaging and therapy agents.
Introduction
purification is done using inline high performance liquid
chromatography which ensures the agents are isolated in high
effective specific activity.
In contrast, the methods used to prepare technetium
radiopharmaceuticals have not changed significantly in over
30 years,3 despite the numerous advantages of technetium-
based agents.4 99mTc is widely available at a low cost, the
radionuclide has excellent nuclear properties for imaging
applications in terms of half-life (6 h) and gamma ray energy
(Eγ ) 140 keV), and it imparts a lower dose burden to
There have been significant advances made recently in the
methods used for the production of positron emission
tomography (PET) agents. Modern technologies such as
automated synthesis modules1 and microfluidic chips2 are
being used to incorporate PET radionuclides, including those
with very short half-lives, into targeting vectors rapidly and
in high yield with minimal handling required. In most cases,
* To whom correspondence should be addressed. E-mail: valliant@
mcmaster.ca.
†
McMaster Nuclear Reactor.
Department of Chemistry.
McMaster Institute of Applied Radiation Sciences (McIARS).
(3) Eckelman, W. C.; Richards, P. J. Nucl. Med. 1970, 11, 761.
(4) (a) Technetium, Rhenium and Other Metals in Chemistry and Nuclear
Medicine, 7; Mazzi, U., Ed.; Servizi Grafici Editoriali snc: Padova,
2006. (b) Technetium and Rhenium in Chemistry and Nuclear
Medicine; Nicolini, M., Bandoli, G., Mazzi, U., Eds.; Raven Press:
New York, 1990. (c) Jurisson, S. S.; Lyden, J. D. Chem. ReV. 1999,
99, 2205. (d) Liu, S.; Edwards, O. S. Chem. ReV. 1999, 99, 2235. (e)
Dilworth, J. R.; Parrott, S. J. Chem. Soc. ReV. 1998, 27, 43. (f) Hom,
R. K.; Katzenellenbogen, J. A. Nucl. Med. Biol. 1997, 24, 485. (g)
Eckelman, W. C. Eur. J. Nucl. Med. 1995, 22, 249. (h) Schwochan,
K. Angew. Chem., Int. Ed. Engl. 1994, 33, 2258.
‡
§
(1) (a) Iwata, R.; Takashashi, M.; Shinohara, M.; Ido, T. J. Labelled Comp.
Radiopharm. 1982, 19, 1350–1351. (b) Alexoff, D. L. In Handbook
of radiopharmaceuticals; Welch, M. J., Redvanly, C. S., Eds.; John
Wiley and Sons Ltd: England, 2003; pp 283-306.
(2) Lee, C.; Sui, G.; Elizarov, A.; Shu, C. J.; Shin, Y. S.; Dooley, A. N.;
Huang, J.; Daridon, A.; Wyatt, P.; Stout, D.; Kolb, H. C.; Witte, O. N.;
Satyamurthy, N.; Heath, J. R.; Phelps, M. E.; Quake, S. R.; Tseng, H.
Science 2005, 310, 1793–1796.
10.1021/ic800775w CCC: $40.75  2008 American Chemical Society
Inorganic Chemistry, Vol. 47, No. 18, 2008 8213
Published on Web 08/19/2008

Causey et al.
patients than most PET radionuclides.5 At present, 99mTc
labeled compounds are produced using single step instant
High resolution mass spectra were obtained on a Waters/Micromass
Global Q-ToF spectrometer. Infrared spectra were obtained on a
BioRad FTS-40 FTIR spectrometer. Purification of all nonradioac-
tive products was achieved by flash chromatography using Ultrapure
Silica Gel from Silicycle (70-230 mesh) or a Biotage SP1 normal
or reversed phase automated purification system. Microwave
reactions were performed using a Biotage Initiator Sixty instrument.
Analytical HPLC was performed using a Varian Pro Star model
330 PDA detector with a model 230 solvent delivery system and a
C-18 Nucleosil column (4.6 × 250 mm) at a flow rate of 1.0 mL
min-1 and monitoring at 254 nm. Specific activity measurements
were performed on an Agilent 1200 series HPLC with an XDB-
C18 column (1.8 µm, 4.6 × 50 mm) at a flow rate of 2.0 mL min-1
and monitoring at 254 nm. The elution protocols used were as
follows: Method A: Solvent A ) methanol, Solvent B ) 0.05 M
TEAP (tetraethylammonium phosphate) buffer (pH 2.2): Gradient
elution 0-3 min, 0% A; 3-6 min 25% A; 6-9 min 33% A; 9-20
min 100% A; 20-22 min 100% A; 22-25 min 0% A; 25-30 min
0% A.15 Method B: Solvent A ) acetonitrile containing 0.1% v/v
TFA, Solvent B ) H2O containing 0.1% TFA v/v: Gradient elution
starting at 5% A to 100% A over 25 min. Method C: Solvent A )
H2O containing 20 mM triethylamine, pH adjusted to 3 with
trifluoroacetic acid, Solvent B ) acetonitrile containing 20 mM
triethylamine, pH adjusted to 3 with trifluoroacetic acid: Gradient
elution starting at 100% A to 60% A over 5 min. Method D: Solvent
A ) H2O containing 20 mM triethylamine, pH adjusted to 3 with
trifluoroacetic acid, Solvent B ) acetonitrile containing 20 mM
triethylamine, pH adjusted to 3 with trifluoroacetic acid: Gradient
elution starting at 70% A to 50% A over 5 min.
A calibration curve for compound 2 ranging in concentration
from 0.02 to 1.0 mM was generated by UV-HPLC using method
B with an internal standard of 2-pyridinecarboxaldehyde (actual
concentrations: 0.017, 0.033, 0.066, 0.133, 0.266, 0.533, 1.066 mM).
Subsequent determination of the limits of quantification (LOQ )
0.062 mM) and limits of detection (LOD ) 0.020 mM) are based
upon the linear regression analysis of the data points which were
each measured in triplicate.
Specific activity for compounds 5b, 6b, and 7b were determined
by generating UV absorbance calibration curves for the corre-
sponding nonradioactive rhenium standards 5a, 6a, and 7a using
HPLC method C for 5b and method D for 6b and 7b. 1-(2-
Methoxyphenyl)-piperazine was used as an internal standard, and
the amount of rhenium standard compound used for linear regres-
sion analyses ranged from 25 ng to 20 000 ng. The signal-to-noise
ratios for all points on the calibration curve were greater than 10.
Samples of approximately 2-5 mCi/100 µL were injected and the
corresponding UV absorbance was translated into mass of sample,
and the collected activity was assayed to determine the final specific
activities. Measurements were done in triplicate.
kits in which the radionuclide as 99mTcO4 is added to a
-
preformulated vial containing a reducing agent, buffer, and
the ligand to be labeled. After mixing the components, the
solution containing the product is typically filtered and
administered directly to patients without any further
purification.
While instant kits are attractive because they provide a
convenient and highly reproducible means of producing Tc
radiopharmaceuticals, they are in fact hindering the develop-
ment of targeted agents. For molecular imaging applications,
the excess ligand that is present in the kit can compete with
the small quantity of labeled product for the target of interest.
Without employing chromatographic methods, this feature
severely limits the range of biological targets for which Tc-
based probes can be developed.6 Furthermore, the perceived
need to use instant kits that have no ancillary purification
methods restricts the choice of targeting vectors to the limited
number of compounds that can be radiolabeled in a single
step in a manner that yields only one product.
To see the impact of such limitations, one need only
consider that if 18F-based PET agents beyond simply fluoride
had to be produced using single step instant kits, there would
be no 18F-based PET tracers in clinical use today. The impact
and widespread use of automated and multistep 18F-synthesis
platforms demonstrates the potential value in modernizing
the way in which Tc radiopharmaceuticals are produced. To
do this will require the development of new rapid multistep
labeling and conjugation methodologies that begin with
pertechnetate and perrhenate in saline and complementary
purification techniques to remove unlabeled ligand that are
all readily automatable. Here, an expedient microwave-based
synthesis platform that can be used to prepare targeted
organometallic Tc(I) and Re(I) bifunctional chelate deriva-
tives in high effective specific activity was developed.
Experimental Section
General Information. Unless otherwise stated, all reactions were
carried out under a nitrogen atmosphere using commercial grade
solvents. Reagents were purchased from Sigma-Aldrich and used
without further purification. Solid-phase extraction (SPE) procedures
were performed using Waters 1.6 mL C18 SepPak cartridges.
Compound 2 was prepared as described in the literature.12
[Re(CO)3(OH2)3]Br and copper-loaded Amberlite IRC-748 resin
were prepared according to literature procedures.13,7 1H and 13C
NMR spectra were recorded on either Bruker AV200, AV600, or
Crystallographic Details. X-ray crystallographic data for 4a
were collected from a sample mounted with epoxy on the end of a
thin glass fiber at 173 K on a Bruker Apex2 Mo diffractometer
(λ ) 0.710 73 Å) with a 3-circle D8 goniometer, fine focus sealed
tube source and graphite monochromator. Data processing was
carried out by use of the program Apex2.8 A numerical face
correction was applied using the Apex2 software. The structure
was solved by using the direct-methods procedure in the Bruker
SHELXTL9 program library and refined by full-matrix least-squares
methods on F2. All non-hydrogen atoms were refined using
1
DRX500 spectrometers. H chemical shifts are reported in ppm
relative to the residual proton signal of the deuterated solvents.
Coupling constants (J) are reported in Hertz (Hz). 13C chemical
shifts are reported in ppm relative to the carbon signal of the solvent.
Low resolution mass spectra were obtained on a Waters/Micromass
Quattro Ultima spectrometer for electrospray ionization experiments.
(5) (a) Mahmood, A.; Jones, A. G. In Handbook of radiopharmaceuticals;
Welch, M. J., Redvanly, C. S., Eds.; John Wiley and Sons Ltd:
England, 2003; pp 323-362. (b) Funk, T.; Sun, M.; Hasegawa, B. H.
Med. Phys. 2004, 31, 2680–2686.
(8) M86-Exx078 APEX2 User Manual; Bruker AXS Inc.: Madison, WI,
2006.
(9) Sheldrick, G. M. SHELXTL, Release 6.14; Siemens Crystallographic
Research Systems: Madison, WI, 2000.
(6) Eckelman, W. C. Nucl. Med. Biol. 2006, 33, 449–451.
(7) Lazarova, N.; James, S.; Babich, J.; Zubieta, Z. Inorg. Chem. Commun.
2004, 7, 1023–1026.
8214 Inorganic Chemistry, Vol. 47, No. 18, 2008

Organometallic Complexes of Tc and Re
anisotropic thermal parameters, and hydrogen atoms were added
as fixed contributors at calculated positions, with isotropic thermal
parameters based on the atom to which they are bonded.
13C NMR (δ (ppm), CD3OD, 150 MHz): 197.18, 196.28, 177.10,
177.01, 162.51, 161.89, 153.08, 141.67, 126.38, 124.52, 71.27,
68.79, 52.47, 36.10, 35.66, 25.88, 23.57, 9.14. IR (KBr, V/cm-1):
2030, 1927, 1702, 1654. HRMS (ES+) (m/z): [M]+ calculated for
[C26H35N5O4Re]+ 668.2221, found 668.2247.
Preparation of [C24H28N4ORe(CO)3]+(6a). The procedure was
the same as that used to prepare 5a. Yield: 27 mg (77%). 1H NMR
(δ (ppm), CDCl3, 200 MHz): 8.64 (d, J ) 4.8 Hz, 2H, PyH), 7.78
(m, 2H, PyH), 7.68 (br s, 1H, NH), 7.50 (m, 2H, PyH), 7.31 (m,
2H, PyH), 7.25 (m, 5H, PhH), 5.34 (d, J ) 16.4 Hz, 2H, PyCH2),
4.48 (m, 2H, PhCH2), 4.31 (d, J ) 16.4 Hz, 2H, PyCH2), 3.66 (m,
2H, NCH2), 2.37 (m, 2H, CH2CO), 1.91 (m, 2H, NCH2CH2), 1.70
(m, 2H, CH2CH2CO). 13C NMR (δ (ppm), CDCl3, 50 MHz):
195.53, 173.12, 160.3, 151.16, 140.44, 138.60, 128.55, 127.85,
127.22, 125.67, 124.00, 70.61, 67.41, 43.45, 35.35, 24.33, 22.75.
IR (KBr, V/cm-1): 2032, 1931, 1685. HRMS (ES+) calcd for
[C27H28N4O4Re]+ 659.1668 (M+), found 659.1642.
Preparation of [C28H35N5O2Re(CO)3]+ (7a). The procedure was
the same as that used to prepare 5a. Yield: 168 mg (82%). 1H NMR
(δ (ppm), CD3OD, 600 MHz): 8.87 (d, J ) 5.6 Hz, 2H, PyH),
7.94 (m, 2H, PyH), 7.55 (d, J ) 7.9 Hz, 2H, PyH), 7.37 (m, 2H,
PyH), 7.04 (m, 1H, ArH), 6.96 (m, 2H, ArH), 6.91 (m, 1H, ArH),
3.87 (s, 3H, OCH3), 3.86 (m, 2H, ReNCH2CH2), 3.77 (t, J ) 5.0
Hz, 2H, NamideCH2CH2N), 3.74 (t, J ) 4.9 Hz, 2H, NamideCH2CH2N),
3.06 (t, J ) 4.9 Hz, NamideCH2CH2N), 3.00 (t, J ) 5 Hz, 2H,
Preparation of [C17H21N3O2Re(CO)3]Br (3a). Compound 2
(0.29 g, 0.97 mmol) and compound 1a (0.65 g, 1.60 mmol) were
combined in a 5 mL microwave vial. A water-acetonitrile mixture
(90:10) was added (3.5 mL) and the reaction vial crimp sealed prior
to heating in the microwave at 150 °C for 5 min with stirring. The
product was purified by loading the reaction solution onto a C18
SepPak cartridge, washing with water (5 mL), and eluting the
desired product with acetonitrile (4 mL). The acetonitrile was
removed under reduced pressure where the resulting amber colored
oil solidified upon standing (0.50 g, 82%). Recrystallization of the
resulting solid from methanol gave colorless crystals of the desired
product. Mp132 °C. 1H NMR (δ (ppm), CD3OD, 600 MHz): 8.87
(d, J ) 5.4 Hz, 2H, PyH), 7.94 (dd, 2H, PyH), 7.56 (d, J ) 7.8 H,
2H, PyH), 7.40 (dd, 2H, PyH), 4.86 (m, 4H, PyCH2), 3.85 (m, 2,
NCH2), 2.47 (t, J ) 7.2 Hz, 2H, CH2CO), 2.01 (m, 2H, NCH2CH2),
1.73 (m, 2H, CH2CH2CO). 13C NMR (δ (ppm), CD3OD, 150 MHz):
197.19, 196.41, 176.91, 162.18, 153.15, 141.60, 126.87, 124.58,
71.55, 68.71, 34.31, 25.63, 23.09. FTIR (KBr, V/cm-1): 2028, 1913,
1739, 1728. HRMS (ES+) calcd for [C20H21N3O5Re]+, 570.1039
(M+); found 570.1027.
Preparation of [C21H24N4O4Re(CO)3]Br (4a). Compound 3a
(0.18 g, 0.28 mmol) was combined with N-hydroxysuccinimide
(0.16 g, 1.4 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbo-
diimide (0.27 g, 1.4 mmol) in a 5 mL microwave vial, and
anhydrous acetonitrile (4 mL) was added. The reaction vial was
crimp sealed, and the mixture heated in the microwave at 120 °C
for 5 min with stirring. The solvent was removed under reduced
pressure, and the desired product isolated by silica gel chromatog-
raphy (2% methanol/98% dichloromethane to 20% methanol/80%
dichloromethane) as a viscous amber oil. The oil solidified upon
standing under ambient conditions, and recrystallization from
methanol gave colorless crystals suitable for X-ray structure
determination (0.19 g, 92%). 1H NMR (δ(ppm), CDCl3, 500 MHz):
8.65 (d, J ) 5.3 Hz, 2H, PyH), 8.01 (m, 2H, PyH), 7.82 (m, 2H,
PyH), 7.20 (m, 2H, PyH), 6.22 (d, J ) 16.2 Hz, 2H, PyCH2), 4.41
(d, J ) 16.2 Hz, 2H, PyCH2), 3.79 (m, 2H, NCH2), 2.84 (s, 4H,
CH2CH2), 2.80, (t, J ) 5.6 Hz, 2H, CH2CO), 2.28 (m, 2H,
NCH2CH2), 1.93 (m, 2H, CH2CH2CO). 13C NMR (δ (ppm), CDCl3,
150 MHz): 195.96, 169.23, 168.26, 161.36, 150.63, 140.34, 125.65,
125.29, 70.54, 67.05, 30.79, 25.79, 24.85, 22.32. FTIR (KBr,
V/cm-1): 2029, 1924, 1735. HRMS (ES+) calcd for
[C24H24N4O7Re]+, 667.1203 (M+); found 667.1224.
N
amideCH2CH2N), 2.61 (t, J ) 7.1 Hz, 2H, CH2C(O)N), 2.01(m,
2H, NCH2CH2), 1.75 (m, 2H, CH2CH2C(O)N); the resonances for
the (PyCH2)2NCH2 protons are obscured by the CD3OD signal. 13
C
NMR (δ (ppm), CD3OD, 150 MHz): 197.22, 196.40, 173.34,
162.20, 153.97, 153.18, 141.97, 141.64, 126.89, 126.34, 125.00,
124.55, 122.21, 119.75, 112.90, 71.54, 71.47, 68.76, 68.47, 56.01,
52.38, 51.89, 47.02, 44.16, 43.08, 33.31, 25.84, 23.36. IR (KBr,
V/cm-1): 2030, 1918, 1686. HRMS (ES+) (m/z): [M]+ calculated
for [C31H35N5O5Re]+ 744.2196, found 744.2191.
Radiochemistry. Caution! 99mTc is a γ-emitter (Eγ ) 140 keV,
t1/2 ) 6 h) while 186Re (Emaxꢀ 1.07 MeV, t1/2 ) 3.8 d) and 188Re
(Emaxꢀ 2.12 MeV, t1/2 ) 17 h) are both ꢀ- and γ-emitters which
should only be used in a licensed and appropriately shielded facility.
Perrhenate solutions (186/188ReO4-; 74-185 MBq) were obtained
from the McMaster Nuclear Reactor following neutron bombard-
ment of naturally occurring rhenium. The average specific activity
of the material was 111 MBq/µg.
-
Preparation of [99mTc(CO)3(OH2)3]+ (1b). 99mTcO4 from a
commercial generator (1600 MBq, 1.5 mL) was added to a crimp
sealed Emry microwave vial (5 mL) containing sodium tartrate (17
mg), sodium tetraborate (3.5 mg), sodium carbonate (3.2 mg), and
potassium boranocarbonate (8.1 mg) under nitrogen. Note that the
reaction vial containing the solid reagents was purged with nitrogen
prior to use for 15 min. The reaction mixture was heated in the
microwave at 130 °C for 3 min with stirring. γ-HPLC (Method
A): tR ) 4 min; Yield: 1600 MBq (quantitative conversion).
Preparation of [C17H21N3O299mTc(CO)3]+ (3b). Compound 2
in distilled water (1 mg/ mL) was added directly to the microwave
vial containing [99mTc(CO)3(OH2)3]+ (1480 MBq), and the reaction
mixture heated in the microwave at 150 °C for 2 min with stirring.
The crude reaction mixture (1221 MBq) was then passed through
a column packed with 400 mg of IRC-748-copper (II) resin (loading
∼1.3 mmol/g) dropwise. Fractions containing the product were
combined and loaded onto a C18 SepPak cartridge, washed with
water (5 mL), and the desired product was eluted with acetonitrile
(4 mL). γ-HPLC (Method A): tR ) 19.2 min; Yield: 943 MBq
(78%, decay corrected).
Preparation of [C23H35N5ORe(CO)3]Br (5a). Compound 4a
(32.3 mg, 0.042 mmol) was dissolved in acetonitrile (1.5 mL) and
transferred to a 5 mL microwave vial. N,N-Diethylethylenediamine
(0.04 mL, 0.28 mmol) was added, the vial crimp sealed, and the
reaction mixture heated in the microwave at 120 °C for 5 min with
stirring. The solvent was removed under reduced pressure, and the
resulting dark oil purified by reverse phase silica gel chromatog-
raphy (95% water/5% acetonitrile to 0% water/100% acetonitrile)
to yield the desired product as a viscous amber oil (25 mg, 80%).
Recrystallization from dichloromethane/diethyl ether gave colorless
1
crystals of the desired product. H NMR (δ (ppm), CD3OD, 600
MHz): 8.86 (d, J ) 5.4 Hz, 2H, PyH), 7.94 (m, 2H, PyH), 7.55 (d,
J ) 7.8 Hz, 2H, PyH), 7.37 (m, 2H, PyH), 3.82 (m, 2H,
NCH2CH2N(Et)2), 3.59 (t, J ) 6.6 Hz, 2H, C(O)NHCH2), 3.28 (m,
6H, N(CH2CH3)2 and NCH2CH2CH2), 2.42 (t, J ) 7.8 Hz,
2H, CH2C(O)), 2.00 (m, 2H, NCH2CH2CH2), 1.74 (m, 2H,
NCH2CH2CH2), 1.34 (t, J ) 7.2 Hz, 6 Hz, CH2CH3); the resonances
for the (PyCH2)2NCH2 protons are obscured by the CD3OD signal.
Inorganic Chemistry, Vol. 47, No. 18, 2008 8215

Causey et al.
Preparation of [C21H24N4O499mTc(CO)3]+ (4b). The acetonitrile
fraction containing compound 3b (666 MBq) was dried using a
Biotage V10 solvent evaporator (T ) 35 °C, 5 min.), the residue
dissolved in anhydrous acetonitrile (4 mL), and the solution
transferred to a 5 mL vial containing N-hydroxysuccinimide (0.16
g, 1.4 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
(0.27 g, 1.4 mmol). The reaction mixture was heated in the
microwave at 120 °C for 5 min with stirring. The product was
purified by loading the reaction mixture onto a C18 SepPak cartridge,
washing with water (5 mL) and eluting the desired product with
acetonitrile (4 mL). γ-HPLC (Method A): tR ) 20 min; Yield: 566
MBq (85%, decay corrected).
mL Emry vial containing N-hydroxysuccinimide (0.20 g, 1.8 mmol)
and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (0.36 g, 1.9
mmol). The reaction mixture was heated in the microwave at 120
°C for 5 min with stirring. The product was purified by loading
the reaction solution onto a C18 SepPak cartridge, washing with
water (5 mL), and eluting the desired product with acetonitrile
(4 mL). γ-HPLC (Method A): tR ) 19.9 min.; Yield: 6.1 MBq
(62%).
Preparation of [C23H35N5O186/188Re(CO)3]+ (5c). N,N-diethyl-
ethylenediamine (18.9 mg, 0.17 mmol) was added to compound
4c (1.9 MBq) in acetonitrile (4 mL) in a 5 mL Emry vial, crimp
sealed, and heated at 120 °C for 5 min with stirring. The product
was verified by comparison of the γ-HPLC with the UV-HPLC of
compound 5a. γ-HPLC (Method A): tR ) 17.1 min.; Yield: 1.9
MBq (>99%).
Preparation of [C23H35N5O99mTc(CO)3]+ (5b). N,N-diethyl-
ethylenediamine (0.04 mL, 0.28 mmol) was added to compound
4b (118 MBq) in acetonitrile (2 mL) in a 5 mL Emry vial, crimp
sealed, and heated at 120 °C for 5 min with stirring. The product
was verified by comparison of the γ-HPLC with the UV-HPLC of
compound 5a. γ-HPLC (Method A): tR ) 17.5 min., Yield: 107
MBq (91%, decay corrected). Specific activity: 4.13 × 108 MBq/
mmol ( 0.02 × 108 MBq/mmol.
Preparation of Compounds [C24H28N4O186/188Re(CO)3]+
(6c). The procedure was the same as that used to prepare 5c. The
product was verified by comparison of the γ-HPLC with the UV-
HPLC of compound 6a. γ-HPLC (Method A): tR ) 20.9 min; Yield:
1.7 MBq (>99%).
Preparation of [C24H28N4O99mTc(CO)3]+ (6b). The procedure
was the same as that used to prepare 5b. The product was verified
by comparison of the γ-HPLC with the UV-HPLC of compound
6a. γ-HPLC (Method A): tR ) 21.2 min; Yield: 118 MBq (91%,
decay corrected). Specific activity: 3.69 × 108 MBq/mmol ( 0.03
× 108 MBq/mmol.
Preparation of [C28H35N5O2186/188Re(CO)3]+ (7c). The proce-
dure was the same as that used to prepare 5c. The product was
verified by comparison of the γ-HPLC with the UV-HPLC of
compound 7a. γ-HPLC (Method A): tR ) 21.9 min; Yield: 2.1
MBq (>99%).
Preparation of [C28H35N5O299mTc(CO)3]+ (7b). The procedure
was the same as that used to prepare 5b. The product was verified
by comparison of the γ-HPLC with the UV-HPLC of the rhenium
standard 7a. γ-HPLC (Method A): tR ) 22.5 min; Yield: 122 MBq
(86%, decay corrected). Specific activity: 4.6 × 108 MBq/mmol (
0.05 × 108 MBq/mmol.
Results and Discussion
One of the challenges in creating a general purpose
platform whereby Tc and Re can be conjugated to a variety
of different targeting vectors is that the product transition
metal complexes must be stable to a wide range of reaction
conditions. Although there has been an elegant in situ
synthesis and purification strategy developed for a bi-
functional Tc(V) chelate system,11 the difficulties of
working in this oxidation state (isomer formation, thermal
stability, etc.) may complicate the use of the technology
as a general purpose platform. With the availability of
the Tc(I)/Re(I) precursor fac-[M(CO)3(OH2)3]+, which can
be prepared in water in a single step,12 it is possible to
prepare inert organometallic complexes with bifunctional
tridentate chelating ligands.13 Taking advantage of the
robust nature of these complexes, a method for the rapid
synthesis and purification of organometallic Re(I) and
Tc(I) complexes, which includes a new method for the
synthesis of the [M(CO)3]+ core, and a variety of
functionalized derivatives was developed.
Microwave Synthesis of [M(CO)3(OH2)3]+ (M ) 99mTc,
186/188Re) and Associated Chelate Complexes. To create a
useful multistep labeling procedure it is essential that all
steps be completed in high yield in as short a period of
time as possible to minimize loss due to decay and to
maximize the specific activity of the product. To achieve
this goal, a series of experiments were performed to
determine if microwave heating could be used to reduce
the amount of time needed to prepare [99mTc(CO)3(OH2)3]+
(1b) and complexation by a bifunctional tridentate ligand
(Scheme 1.) To this end, pertechnetate was added to a 5
Preparation of [186/188Re(CO)3(OH2)3]+ (1c). ortho-Phosphoric
acid (71 µL) was added to high purity water (medical grade, 5 mL),
and the solution was degassed with N2 for 15 min. An aqueous
-
solution (0.5 mL) containing 186/188ReO4 (42 MBq) was added,
and an aliquot (1.5 mL) of that solution added to a sealed Emry
vial containing sodium tartrate (16 mg), sodium tetraborate (4 mg),
sodium carbonate (3.2 mg), sodium boranocarbonate (8.6 mg), and
BH3 ·NH3 (17 mg) under nitrogen. Gas evolution was observed,
and the solution turned amber in color. The reaction mixture was
heated in a microwave at 150 °C for 3 min with stirring. The product
was compared by γ-HPLC to the desired product synthesized
according to the Mallinckrodt two-step kit formulation.10 γ-HPLC
(Method A): tR ) 3.5 min. Yield: 39.2 MBq (93%).
Preparation of [C17H21N3O2186/188Re(CO)3]+ (3c). Compound
2 (0.29 g, 0.97 mmol) dissolved in degassed water for injection
(WFI, 0.5 mL) was added directly to an aqueous solution of
compound 1c (77 MBq) in a crimp sealed Emry microwave vial,
and the mixture heated in the microwave at 150 °C for 2 min with
stirring. The product was purified by loading the reaction solution
onto a C18 SepPak cartridge, washing with water (5 mL) and then
acetonitrile (4 mL). γ-HPLC (Method A): tR ) 19.1 min; Yield:
68 MBq (90%).
Preparation of [C21H24N4O4186/188Re(CO)3]+ (4c). The aceto-
nitrile solution containing compound 3c (9.9 MBq) was evaporated
to dryness using a Biotage V10 solvent evaporator. Anhydrous
acetonitrile (4 mL) was added and the mixture transferred to a 5
(10) A two-component kit was kindly supplied by Mallinckrodt Medical,
Inc. The kit was labeled with rhenium according to instructions
supplied by the manufacturer. The radiochemical purity of the material
was determined using g-HPLC to assess the percentage of free
perrhenate.
(11) Misra, P.; Humblet, V.; Pannier, N.; Maison, W.; Frangioni, J. V.
J. Nucl. Med. 2007, 48, 1381–1389.
8216 Inorganic Chemistry, Vol. 47, No. 18, 2008

Organometallic Complexes of Tc and Re
Scheme 1. Expedient Synthesis of M(CO)3-Bifunctional Chelate Complexesa
a
1. 130 °C, 5 min (M ) Re), 130 °C, 3 min (M ) 99mTc) or 150 °C, 3 min (M ) 186/188Re). 2. 150 °C, 5 min (M ) Re) or 150 °C, 2 min (M ) 99mTc
and 186/188Re). 3. 120 °C, 5 min, N-hydroxysuccinimide, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide. 4. 120 °C, 5 min, N,N-diethylethylenediamine,
benzylamine, or 1-(2-methoxy)-phenylpiperazine.
Figure 1. γ-HPLC chromatogram of 1b produced by microwave (top) (T ) 130 °C, time ) 3 min) and conventional (bottom) heating (T ) 95 °C, time
) 30 min) (HPLC method A).
mL microwave vial containing the same components
traditionally used to prepare 1b (sodium-boranocarbonate,
Na/K tartrate, sodium tetraborate decahydrate, and sodium
carbonate), and the mixture heated in a microwave at a
range of different temperatures for varying amounts of
time. Radio-HPLC analyses of the resulting mixtures
showed that the optimal conditions were 130 °C for 3 min
and that the product was the same as the material produced
using conventional heating (Figure 1.) At temperatures
-
above 130 °C, degradation of the desired product to TcO4
occurred. By heating at 130 °C however, 1b was prepared
with exquisite reproducibility in nearly quantitative yield
Inorganic Chemistry, Vol. 47, No. 18, 2008 8217

Causey et al.
nonradioactive Re analogue 3a (Figure 3.)
One of the limitations of using the tricarbonyl core for
developing molecular imaging agents, particularly for targets
that are expressed in low concentration, is that a substantial
amount of ligand typically must be used during labeling
reactions to achieve good radiochemical yields. This feature
limits the maximum achievable specific activity unless
ancillary purification methods like HPLC are employed. The
impact of microwave heating on the radiochemical yield as
a function of ligand concentration was tested to see if it was
possible to reduce the amount of ligand required in a given
reaction. At 10-3 M ligand concentrations, quantitative yields
could be achieved and even at levels as low as 10-6 M some
complex formation was observed (11%). This is in contrast
to conventional heating at this level which produced primarily
pertechnetate and unreacted starting material. Below a
concentration of 10-6 M, the yield decreased dramatically,
even with prolonged microwave heating.
Figure 2. γ-HPLC chromatogram of 1c formed by microwave heating
(HPLC method A).
in mere minutes as opposed to greater than 20 min using
conventional methods.8
The excellent yields and reduced synthesis times obtained
for the production of 1b lead us to investigate the synthesis
of the radioactive rhenium analogues (i.e., 186/188Re) (Scheme
1). The current method used to prepare [186/188Re-
(CO)3(OH2)3]+ 1c involves the use of a two-step kit because
Purification via Chemoselective Filtration. At 10-3
M
ligand concentrations there was still an appreciable amount
of unlabeled 2 remaining in solution. This material can be
removed by semipreparative HPLC; however, such an
approach is not desirable for producing agents that are
destined to be used clinically or for an automated synthesis
platform. To provide a means of producing in high effective
specific activity, a convenient resin capture purification
method was developed which takes advantage of the reactiv-
ity of the unlabeled chelate. The system is based on a simple
copper functionalized solid-support which was used by Ley
et al. to isolate bipyridyl derivatives of small molecules from
reaction mixtures via coordination to the resin.17
Copper-loaded Amberlite IRC-748 resin (loading ∼1.3
mmol/g) was prepared according to a literature procedure,18
and a solution containing 2 and 3b (1221 MBq) was passed
through a syringe filled with approximately 400 mg of resin
(Scheme 2.) To test the efficiency of the purification strategy
(Figure 4,) a large excess of the ligand was employed during
the labeling reaction (1.55 mM). After treatment with the
copper resin, the amount of residual ligand was less than
the detection limit of the HPLC (0.020 mM). For some
batches of resin, formation of the Cu complex of 2 was
detected by HPLC (verified through comparison to an
authentic standard) which we believe is due to incomplete
removal of free copper ion during the preparation of the resin.
Notwithstanding, the copper complex if formed can be easily
separated from the desired product using a C18 SPE
cartridge, which is a procedure routinely used in automated
radiochemistry systems. The average amount of radioactivity
recovered was 78% across all experiments. This modest loss
of activity is acceptable given the speed of the overall
procedure, the exquisite purity of the product, and the low
cost of 99mTc.
-
of the greater reduction potential of ReO4 compared to
14,15
-
TcO4 .
To produce the desired product following the
conventional method, it is necessary to pretreat ReO4- with
phosphoric acid and BH3 · NH3 prior to performing the
reductive carbonylation reaction using the Isolink kit. With
microwave heating, the desired product can be produced in
a single step by adding BH3 · NH3 and phosphoric acid to
the formulation used to prepare 1b. Heating the reaction
mixture at 150 °C for 3 min produced 1c in close to
quantitative yield (Figure 2.) The ability to produce [186/188Re-
(CO)3(OH2)3]+ in a single pot is advantageous because it
greatly decreases the overall reaction time and reduces the
amount of sample handling required.
In addition to using microwave heating to prepare
[M(CO)3(OH2)3]+ (1b and 1c), we also explored its use in
promoting metal complexation with a bifunctional chelating
agent (Scheme 1). The ligand selected, which was developed
by Zubieta and co-workers,16 contains two pyridine groups
and one tertiary amine which form a stable cationic complex
with the tricarbonyl core. Labeling reactions were performed
by direct addition of compound 2 in solution to the
microwave vial containing 1b or 1c. Through a temperature
course study, it was determined that quantitative conversion
to the desired metal complex was achieved in less than two
minutes at 150 °C. The identities of the metal complexes
3b and 3c were verified by comparison of the γ-HPLC traces
of the products with UV-HPLC traces of fully characterized
(12) Alberto, R.; Ortner, K.; Wheatley, N.; Schibli, R.; Schubiger, A. P.
J. Am. Chem. Soc. 2001, 123, 3135–3136.
(13) Banerjee, S. R.; Maresca, K. P.; Francesconi, L.; Valliant, J.; Babich,
J. W.; Zubieta, J. Nucl. Med. Biol. 2005, 32, 1–20.
(14) Schibli, R.; Schwarzbach, R.; Alberto, R.; Ortner, K.; Schmalle, H.;
Dumas, C.; Egli, A.; Schubiger, P. A. Bioconjugate Chem 2002, 13,
750–756.
Rapid Evaporation and Activation. The next step was
(15) Park, S. H.; Seifert, S.; Pietzsch, H. J. Bioconjugate Chem. 2006, 17,
223–225.
(17) Ley, S. V.; Massi, A.; Rodriguez, F.; Horwell, D. C.; Lewthwaite,
R. A.; Pritchard, M. C.; Reid, A. M. Angew. Chem., Int. Ed. 2001,
40, 1053–1055.
(16) Banerjee, S. R.; Levadala, M. K.; Lazarova, N.; Wei, L.; Valliant,
J. F.; Stephenson, K. A.; Babich, J. W.; Maresca, K. P.; Zubieta, J.
Inorg. Chem. 2002, 41, 6417–6425.
(18) Siu, J.; Baxendale, I. R.; Lewthwaite, R. A.; Ley, S. V. Org. Biomol.
Chem. 2005, 3, 3140–3160.
8218 Inorganic Chemistry, Vol. 47, No. 18, 2008

Organometallic Complexes of Tc and Re
Figure 3. UV-HPLC chromatogram of crude reaction mixture for the synthesis of 3b showing excess 2 along with co-injected 3a (top) and the γ-HPLC
chromatogram of the reaction mixture containing 3b (bottom).
Scheme 2. Removal of Unreacted Ligand 2 by Chemoselective Filtration
to develop a convenient means to link the purified chelate
complexes to targeting vectors. The approach taken involved
conversion of the pendent acid group to an active ester so
that the chelate complexes can be conjugated to amino
groups. Generally, the preparation of active esters is best
performed in organic solvents. This is not an option for
traditional Tc instant kits as they are designed to work with
°C. Analysis of the product following the evaporation
procedure showed no signs of degradation which is consistent
with the mild nature of the drying process and the known
stability of the complex. If high effective specific activity is
not a critical issue, the copper resin procedure can be omitted
and the aqueous solution containing the metal complex
concentrated directly following microwave heating.
Microwave heating was subsequently used to produce the
active ester 4b. Anhydrous acetonitrile was added to dried
3b in a 5 mL microwave vial along with N-hydroxysuccin-
imide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
(EDC). The reaction mixture was heated at 120 °C for 5
min, and the product isolated by loading the reaction solution
onto a C18 SepPak cartridge, washing with water (5 mL),
and eluting the desired product with acetonitrile (4 mL). The
identity of 4b, which was isolated in 85% yield, was verified
by comparison of the γ-HPLC trace with the UV-HPLC trace
of the rhenium standard 4a. The nonradioactive Re analogue
was also prepared in the microwave in the same manner used
for the Tc complex and was isolated in high yield (92%).
An X-ray crystal structure of the product was obtained
the 0.9% saline solution used to elute 99mTcO4 from the
-
99Mo/99mTc generators. In contrast, for PET chemistry,
organic solvents are used routinely to accelerate labeling
reactions and to be able to utilize reactive compounds that
would hydrolyze in aqueous solutions.
Following removal of unlabeled ligand using the copper
resin, the aqueous solution containing 3b was loaded onto a
C18 SepPak cartridge which was subsequently washed with
water (5 mL) to remove salts and then acetonitrile (4 mL)
to elute the desired product. The acetonitrile fraction contain-
ing compound 3b (666 MBq) was dried using a Biotage V10
solvent evaporator which is a new technology for rapid
removal of solvents under mild conditions. The evaporation
was complete in less than five minutes with heating at 35
Inorganic Chemistry, Vol. 47, No. 18, 2008 8219

Causey et al.
Table 1. Crystal and Structure Refinement Data for 4a
empirical formula
formula weight
temperature
C24H26BrN4O8Re
764.60
173(2) K
crystal system
space group
monoclinic
P2(1)/c
unit cell dimensions
a ) 9.0064(6) Å
b ) 14.7027(11) Å, ꢀ ) 91.7070(10)°
c ) 20.1519(15) Å
Z
4
goodness-of-fit on F2
final R indices [I > 2σ(I)]
R indices (all data)
1.430
R1 ) 0.0513, wR2 ) 0.1929
R1 ) 0.0593, wR2 ) 0.2050
Table 2. Select Bond Lengths and Angles for Compound 4a
Re(1)-C(2)
Re(1)-C(3)
Re(1)-C(1)
Re(1)-N(3)
Re(1)-N(2)
Re(1)-N(1)
N(4A)-O(5A)
1.915(9)
1.916(8)
1.918(9)
2.181(7)
2.192(6)
2.240(6)
C(2)-Re(1)-C(3)
C(2)-Re(1)-C(1)
C(3)-Re(1)-C(1)
C(2)-Re(1)-N(3)
C(3)-Re(1)-N(3)
C(1)-Re(1)-N(3)
87.8(4)
89.3(4)
85.3(4)
95.9(3)
97.9(3)
174.0(3)
171.9(3)
96.2(3)
98.0(3)
76.6(2)
97.5(3)
173.3(3)
98.9(3)
77.5(2)
78.1(2)
113.9(10)
1.379(13) C(2)-Re(1)-N(2)
O(4A)-C(20A) 1.210(14) C(3)-Re(1)-N(2)
O(5A)-C(20A) 1.372(14) C(1)-Re(1)-N(2)
Figure 4. γ-HPLC (top) and UV-HPLC (bottom) chromatograms of 3b
after treatment with the copper resin.
N(4)-O(5)
N(4)-C(21)
N(4)-C(24)
O(4)-C(20)
O(5)-C(20)
O(6)-C(21)
1.383(14) N(3)-Re(1)-N(2)
1.404(18) C(2)-Re(1)-N(1)
1.488(18) C(3)-Re(1)-N(1)
1.211(16) C(1)-Re(1)-N(1)
1.380(14) N(3)-Re(1)-N(1)
1.184(16) N(2)-Re(1)-N(1)
C(20A)-O(5A)-N(4A)
C(24A)-O(7A)-H(7AA) 109.5
O(4A)-C(20A)-O(5A)
O(6A)-C(21A)-N(4A)
O(5)-N(4)-C(21)
122.3(11)
118.8(16)
122.5(12)
120.8(11)
110.1(12)
112.0(11)
O(5)-N(4)-C(24)
C(21)-N(4)-C(24)
C(20)-O(5)-N(4)
istration to animal models. It is clear from these experi-
ments that the active ester is sufficiently reactive even at
high effective specific activity to efficiently couple with
a variety of different amines. The typical specific activity
of the 99mTc-labeled amides produced using this strategy
was between 3.7 × 108 MBq/mmol and 4.6 × 108 MBq/
mmol, in close agreement with a previously reported
specific activity values for multistep labeling of small
molecule radiotracers (4.1 × 108 MBq/mmol).11
Conclusions
An indirect labeling method for preparing organometallic
Tc and Re chelate complexes was developed. The process
which involved microwave heating, in situ purification, and
rapid solvent evaporation was efficient, high yielding, and
produced the desired products in high effective specific
activity. The entire synthetic procedure, which can be easily
converted into an automated synthesis platform, was com-
pleted in less than 30 min, and the products were generated
in high effective specific activity. Performing the same series
of reactions using conventional heating and evaporation
methods required greater than 2 h, and the decay corrected
yields were not as high (57%) as those obtained using the
microwave-V10 evaporator system (86%). The approach
described here represents a movement away from conven-
tional instant kits toward automated multistep synthesis and
purification platforms which should help accelerate the
process of discovering novel 99mTc and Re molecular imaging
Figure 5. X-ray crystal structure of 4a (30% thermal probability ellipsoids).
The bromide anion and a water molecule were removed for clarity.
following recrystallization from methanol (Figure 5, Tables
1 and 2).
The reactivity of the active esters 4b and 4c were tested
using a series of model amines including N,N-diethyleth-
ylenediamine, benzylamine, and 1-(2-methoxy)-phenylpip-
erazine (Scheme 1). Reactions were performed in the
microwave at 120 °C for 5 min. Yields were typically
greater than 90% and the products isolated by SPE. In
the case of complex 4c, reaction with a secondary amine
produced the desired product 7b in somewhat lower yield
than for the technetium analogue but still acceptable at
86%. The reaction solvent was readily removed using the
V10 evaporator so that the final products could be
reconstituted in saline solutions suitable for direct admin-
8220 Inorganic Chemistry, Vol. 47, No. 18, 2008

Organometallic Complexes of Tc and Re
and therapy agents and increase the number of new targeted
agents entering the clinic. The technique will also be
applicable for use with other nonstandard radionuclides such
as the PET isotope 94mTc which has a relatively short half-
life (t1/2 ) 54 min).
the MAX Diffraction Facility at McMaster University for
performing the X-ray analyses and preparing the structure
for publication.
Supporting Information Available: Complete spectral data for
all novel Re compounds and relevant HPLC chromatograms for
the radiolabeled compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by funding
from the Canadian Institutes for Health Research (CIHR),
the Ontario Institute of Cancer Research, and the Cancer
Imaging Network of Ontario (CINO). We thank the staff of
IC800775W
Inorganic Chemistry, Vol. 47, No. 18, 2008 8221