Acyclic chelate with ideal properties for 68Ga PET imaging agent elaboration

Article abstract of DOI:10.1021/ja106399h

We have investigated novel bifunctional chelate alternatives to the aminocarboxylate macrocycles NOTA (N₃O₃) or DOTA (N ₄O₄) for application of radioisotopes of Ga to diagnostic nuclear medicine and have found t

Full text of DOI:10.1021/ja106399h

Published on Web 10/19/2010
Acyclic Chelate with Ideal Properties for 68Ga PET Imaging
Agent Elaboration
Eszter Boros,†,§ Cara L. Ferreira,‡ Jacqueline F. Cawthray,†,§ Eric W. Price,†
Brian O. Patrick,† Dennis W. Wester,‡ Michael J. Adam,*,§ and Chris Orvig*,†
Medicinal Inorganic Chemistry Group, Department of Chemistry, UniVersity of British
Columbia, 2036 Main Mall, VancouVer, British Columbia V6T 1Z1, Canada, MDS Nordion,
4004 Wesbrook Mall, VancouVer, British Columbia, Canada V6T 2A3, and TRIUMF,
4004 Wesbrook Mall, VancouVer, British Columbia, Canada V6T 2A3
Received July 27, 2010; E-mail: adam@triumf.ca; orvig@chem.ubc.ca
Abstract: We have investigated novel bifunctional chelate alternatives to the aminocarboxylate macrocycles
NOTA (N3O3) or DOTA (N4O4) for application of radioisotopes of Ga to diagnostic nuclear medicine and
have found that the linear N4O2 chelate H2dedpa coordinates 67Ga quantitatively to form [67Ga(dedpa)]+
after 10 min at RT. Concentration-dependent coordination to H2dedpa of either 68Ga or 67Ga showed
quantitative conversion to the desired products with ligand concentrations as low as 10-7 M. With 68Ga,
specific activities as high as 9.8 mCi nmol-1 were obtained without purification. In a 2 h competition
experiment against human apo-transferrin, [67Ga(dedpa)]+ showed no decomposition. Two bifunctional
versions of H2dedpa are also described, and these both coordinate to 67Ga at RT within 10 min. Complete
syntheses, characterizations, labeling studies, and biodistribution profiles of the 67Ga complexes are
presented for the new platform chelates. The stability of these platform chelates is higher than that of
DOTA.
Introduction
The efficient and strong chelation of this radiometal in its
only biologically stable oxidation state 3+ has been investigated
for the past 40 years with high hopes for potentially useful
complexes in radiopharmacy.8,9 The sustained efforts of Martell
and Welch over several decades2,10 have been succeeded by
the significant progress of Maecke and co-workers in the late
1990s when bifunctional versions of the tri- and tetra-aza-based
aminocarboxylate macrocyclic chelators NOTA and DOTA were
able to deliver highly promising results not only for the isotopes
68Ga and 111In but also for a wide range of radiolanthanides.11
Many attempts to find chelators of comparable kinetic and
thermodynamic stability for 68Ga have been less successful.12-14
The macrocyclic chelators themselves are challenging to
Disruptions in the supply chain of 99Mo, the parent of the
clinically important daughter isotope 99mTc, have worldwide
effects on diagnostic nuclear medicine and have turned many
researchers’ attention to other generator-produced isotopes.1
Millions of diagnostic procedures annually take advantage of
the 99Mo/99mTc isotope pair for single-photon-emission computed
tomography (SPECT); Canada, among other countries, has in
recent times been responsible for the interrupted supply chain.
Among attractive alternatives to 99Mo/99mTc is the 68Ge/68Ga
generator system,2 which has the potential to be eluted for up
to one year due to the long half-life (t1/2 ) 271 d) of the parent
radionuclide 68Ge. The 68Ga generator has been used to prepare
68Ga radiopharmaceuticals for clinical imaging in Europe for
several years,3 but the development of a generator with
regulatory approval for human use would further facilitate the
transition of 68Ga based agents into the clinic in North America.
Ga forms stable complexes with many multidentate ligands,4
and the daughter radionuclide, 68Ga, has suitable properties for
high-quality positron-emission tomography (PET) imaging
including a short half-life (t1/2 ) 68 min), decay by 89% positron
emission and a maximum positron energy of 1.899 keV.5-7
(6) Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J. Chem.
ReV. 2010, 110, 2858–2902.
(7) Bartholoma, M. D.; Louie, A. S.; Valliant, J. F.; Zubieta, J. Chem.
ReV. 2010, 110, 2903–2920.
(8) Maecke, H. R.; Andre´, J. P. 68Ga-PET radiopharmacy: A generator-
based alternative to 18F-radiopharmacy. In Ernst Schering Research
Foundation Workshop; Springer: New York, 2007; Vol. 62, pp 215-
242.
(9) Anger, H. O.; Gottschalk, A. J. Nucl. Med. 1963, 4, 326.
(10) Jones-Wilson, T. M.; Motekaitis, R. J.; Sun, Y.; Anderson, C. J.;
Martell, A. E.; Welch, M. J. Nucl. Med. Biol. 1995, 22 (7), 859–68.
(11) Andre, J. P.; Maecke, H. R.; Zehnder, M.; Macko, L.; Akyel, K. G.
Chem. Commun. 1998, 12, 1301–1302.
† University of British Columbia.
‡ MDS Nordion.
(12) Bollinger, J. E.; Mague, J. T.; O’Connor, C. J.; Banks, W. A.;
Roundhill, D. M. Dalton Trans. 1995, 10, 1677–1688.
(13) Mathias, C. J.; Lewis, M. R.; Reichert, D. E.; Laforest, R.; Sharp,
T. L.; Lewis, J. S.; Yang, Z.-F.; Waters, D. J.; Snyder, P. W.; Low,
P. S.; Welch, M. J.; Green, M. A. Nucl. Med. Biol. 2003, 30, 725–
731.
§ TRIUMF.
(1) Green, M. A. J. Nucl. Med. 1990, 31, 1641–1645.
(2) Gleason, G. I. Int. J. Appl. Radiat. Isot. 1960, 8, 90–94.
(3) Rufini, V.; Calcagni, M. L.; Baum, R. P. Semin. Nucl. Med. 2006, 36,
228–247.
(4) Harris, W. R.; Martell, A. E. Inorg. Chem. 1976, 15, 713–720.
(5) Bandoli, G.; Dolmella, A.; Tisato, F.; Porchia, M.; Refosco, F. Coord.
Chem. ReV. 2009, 253, 56–77.
(14) Cutler, C. S.; Giron, M. C.; Reichert, D. E.; Snyder, A. Z.; Herrero,
P.; Anderson, C. J.; Quarless, D. A.; Koch, S. A.; Welch, M. J. Nucl.
Med. Biol. 1999, 30, 305–316.
9
15726 J. AM. CHEM. SOC. 2010, 132, 15726–15733
10.1021/ja106399h  2010 American Chemical Society

67/68Ga Complexes of Acyclic Chelates
A R T I C L E S
Scheme 1. NOTA, DOTA, H2dedpa, and dedpa Derivatives, 3 and 7
these chelates using this generator have been reported.16 Analysis
of radiolabeled complexes was done on a Phenomenex Hydrosyn-
ergy RP C18 4.6 mm × 150 mm analytical column ([Ga(dedpa)]+),
Phenomenex Jupiter 5µ C18 300 A 4.6 mm × 100 mm (transferrin
(Tf) challenge with [Ga(dedpa)]+, NOTA Versus H2dedpa challenge,
H2dedpa challenge of Ga(NOTA), retention time of 67Ga-Tf: 10.7
min) and Waters XBridge BEH130 4.6 mm × 150 mm ([Ga3]+,
[Ga7]+, as well as the transferrin challenges thereof, retention time
of 67Ga-Tf: 2.5 min).
synthesize as selectively functionalized analogues, and most
research groups in need of bifunctional derivatives of NOTA
or DOTA are supplied by a commercial source.
Herein we report a linear chelating ligand with optimal
properties for the field of gallium radiopharmaceutical chemistry;
H2dedpa forms stable complexes of Ga quickly and under mild
conditions, lending itself well to elaboration with targeting
vectors for radionuclide delivery. Initial studies of dedpa2-
coordination with Ga and two of its radioisotopes 67,68Ga, as
well as the preparation of bioconjugate precursors 3 and 7
(Scheme 1), are reported along with relevant transferrin
competition studies and biodistribution results. These most
promising results suggest that dedpa2- and its derivatives have
great potential to be powerful new tools for the radiopharma-
ceutical chemistry of gallium.
H2dedpa ·2HCl. Protected precursor 1,2-[{6-(methoxycarbon-
yl)pyridin-2-yl}methylamino]ethane was synthesized according to
the literature.17 Deprotection of 1,2-[{6-(methoxycarbonyl)-pyridin-
2-yl}methylamino]ethane was achieved by dissolving (37 mg, 0.1
mmol) in 4 mL of a 1:1 mixture of THF and water. LiOH (10 mg,
0.41 mmol, 4.1 equiv) was added and the reaction mixture was
stirred for 2.5 h. Reaction monitoring was performed by TLC (20%
CH3OH in CH2Cl2, tR of starting material: 0.8, tR of product: 0.0).
The solvent was removed in Vacuo, and 12 M HCl was added to
the glass-like solid to precipitate the dihydrochloride salt, which
was collected by filtration to afford 24 mg (0.059 mmol, 59%) of
Experimental Section
Materials and Methods. All solvents and reagents were from
commercial sources and were used as received unless otherwise
indicated. Human serum apo-transferrin was purchased from Sigma-
Aldrich (St. Louis, MO). The analytical thin-layer chromatography
(TLC) plates were aluminum-backed ultrapure silica gel 60, 250
µm; the flash column silica gel (standard grade, 60 Å, 32-63 mm)
was provided by Silicycle. 1H and 13C NMR spectra were recorded
at RT on Bruker AV300, AV400, or AV600 instruments; the NMR
spectra are expressed on the δ scale and were referenced to residual
solvent peaks or internal tetramethylsilane. Electrospray-ionization
mass spectrometry (ESI-MS) spectra were recorded on a Micromass
LCT instrument at the Department of Chemistry, University of
British Columbia. IR spectra were collected neat in the solid state
on a Thermo Nicolet 6700 FT-IR spectrometer. The HPLC system
used for analysis consisted of a Waters Alliance HT 2795 separation
module equipped with a Raytest Gabi Star NaI (Tl) detector and a
Waters 996 photodiode array (PDA) detector. 67Ga was obtained
as a 0.1 M HCl solution, and 68Ga (5-10 mCi/mL) (both MDS
Nordion Inc.) was obtained from a generator constructed of titanium
dioxide sorbent that was charged with 68Ge and eluted with aqueous
HCl (0.1 M).15 The generator has been previously used for
radiolabeling NOTA- and DOTA-based chelate systems, and the
resulting radiochemical yields and specific activities achievable for
1
a white solid. H NMR (d6-DMSO, 300 MHz) δ: 8.12-8.09 (m,
4H, ortho-/para-H), 7.78 (d, ortho-H), 4.52 (s, 2H, CH2), 3.51 (d,
2H, CH2). 13C NMR (d6-DMSO, 150 MHz) δ: 166.1, 153.5, 148.1,
139.7, 127.2, 124.9, 104.6, 93.8, 50.7, 43.8. IR (cm-1): 2678, 2600,
2427, 1761, 1749, 1599. HR-ESI-MS calcd for C16H19N4O4:
331.1406; found: 331.1329 [M+H]+. Elemental analysis: calcd %
for H2dedpa ·2HCl (402.8): C 47.65, H 5.00, N 13.81; found: C
47.30, H 5.11, N 13.38.
[Ga(dedpa)][ClO4]. H2dedpa ·2HCl (21 mg, 0.052 mmol) was
dissolved in a CH3OH-water mixture (1:2). Ga(ClO4)3 ·6H2O (24
mg, 0.052 mmol) was added, and the pH was adjusted to 4.5 by
addition of 0.1 M NaOH. The reaction mixture was heated for 30
min and then set aside in the fume hood for slow evaporation. After
72 h, rhombic colorless crystals suitable for X-ray diffraction had
precipitated in quantitative yield. 1H NMR (300 MHz, d6-DMSO)
δ: 8.59 (t, 2H, para-H), 8.29 (d, 2H, meta-H), 8.09 (d, meta-H),
4.60-4.32 (dd, 4H, py-CH2-NH), 3.06 (m, 2H, CH2), 2.4 (m, 2H,
CH2). 13C NMR (75 MHz, d6-DMSO) δ: 162.0, 150.4, 145.2, 144.1,
(16) Ferreira, C. L.; Lamsa, E.; Woods, M.; Duan, Y.; Fernando, P.;
Bensimon, C.; Kordos, M.; Guenther, K.; Jurek, P.; Kiefer, G. E.
Bioconjugate Chem. 2010, 21 (3), 531–536.
(17) Platas-Iglesias, C.; Marto-Iglesias, M.; Djanashvili, K.; Muller, R. N.;
Vander Elst, L.; Peters, J. A.; de Blas, A.; Rodrigues-Blas, T.
Chem.sEur. J. 2004, 3579, 3590.
(15) Malyshev, K. V.; Smirnov, V. V. Radiokhimiya 1975, 17, 137–140.
9
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A R T I C L E S
Boros et al.
129.2, 126.5, 122.0. HR-ESI-MS calcd for C16H1669GaN4O4:
397.0427; found: 397.0431 [M]+. IR (cm-1): 2360, 2341, 1695,
1664, 1606. Product tR on HPLC: 5.5 min (gradient: A - NaOAc
buffer, pH 4.5; B - MeOH, 0-5% B linear gradient 20 min).
benzyl-H), 8.18 (d, 2H, py-H), 7.72 (d, 2H, benzyl-H), 5.03-4.34
(dd, 4H, py-CH2-NH), 4.18-3.87 (dd, 4H, -CH2-CH2-), 3.13 (s (br),
4H, benzyl-CH2-NH). 13C NMR (150 MHz, CD3OD) δ: 165.1,
152.0, 150.3, 148.3, 145.7, 138.1, 134.5, 129.8, 125.6, 124.8, 57.7,
55.4, 48.1. HR-ESI-MS calcd. for C30H26N6O869Ga: 667.1068;
found: 667.1075 [69 M]+.
[
67/68Ga(dedpa)]+. General Labeling Procedure. 67GaCl3 (100
µL, 1 mCi) or 68Ga3+ in a 0.1 M HCl solution was added to 900
µL of a 10-4 M solution of ligand in 10 mM NaOAc solution (pH
4.5) and left for 10 min at RT. The reaction progress was monitored
by analytical HPLC which showed that the reaction had proceeded
to 99%. Product tR on HPLC: 6.1 min (gradient: A - NaOAc buffer,
pH 4.5; B - CH3OH, 0-5% B linear gradient 20 min). The high
specific activity of 9.837 ( 0.136 mCi/nmol, with yields 99.9 (
0.1% was achieved with 900 µL of a 10-7 M solution of H2dedpa
in 10 mM NaOAc solution (pH 4.5) and 100 µL 68Ga3+ in a 0.1 M
HCl solution (0.98 mCi) under standard labeling conditions (as
described above); this experiment was done in triplicate.
Complex Stability against Transferrin. For apo-transferrin
competition, 67GaCl3 was added to a 10-4 M solution of ligand in
10 mM NaOAc solution (pH 4.5). Complex formation was checked
by HPLC. A 400-µL aliquot was added to 1 mg/mL apo-transferrin
in a NaHCO3 solution (10 mM, 600 µL) and incubated at 37 °C
(water bath). Complex stability was checked at time points 10 min,
1 h, and 2 h via analytical HPLC. No decomposition was detected.
Competition for Chelation Experiment with NOTA. 67GaCl3
was added to 10-4 M solution of both NOTA and H2dedpa in 10
mM NaOAc solution (pH 4.5). After a reaction time of 10 min at
room temperature the reaction mixture was checked for the formed
complex by analytical HPLC. Over 98% of the 67Ga-dedpa complex
was detected, opposed to 0.2% Ga-NOTA.
[
67/68Ga(3)]+. 67GaCl3 (100 µL, 1 mCi) or 68Ga3+ ( 100 µL, 1
mCi) in a 0.1 M HCl solution was added into 10-4 M solution of
ligand in 10 mM NaOAc solution (pH 4) and left to react for 10
min at room temperature. Reaction control was performed by
analytical HPLC which showed that the reaction had proceeded to
98%. Product tR on HPLC: 10.8 min (gradient: A - NaOAc buffer,
pH 4.5; B - CH3OH, 0-100% B linear gradient 20 min). For the
apo-transferrin competition, 67GaCl3 was added to 10-4 M solution
of 3 in 10 mM NaOAc solution (pH 4.5). Complex formation was
checked on HPLC (peptide column). A 400-µL aliquot was added
to 1 mg/mL apo-transferrin in a NaHCO3 solution (10 mM, 600
µL) and incubated at 37 °C (water bath). Complex stability was
checked at time points 10 min, 1 h, and 2 h via analytical HPLC.
Complex was 51% intact after 2 h.
2-(p-Nitrobenzyl)-N,N′-[6-{methoxycarbonyl}pyridin-2-yl]me-
thylamino)ethane (6). 4 and 5 were synthesized according to the
literature.17,18 To a mixture of 4 (0.46 g, 2.36 mmol) in methanol
(50 mL), was added 5 (0.78 g, 4.72 mmol). The mixture was
refluxed for 2 h and then cooled to 0 °C in an ice bath. After
cooling, NaBH4 (0.139 g, 3.67 mmol) was added slowly and stirred
at 0 °C for 2 h. Saturated aqueous NaHCO3 was then added (150
mL) and the mixture stirred for 15 min, followed by extraction
with dichloromethane (5 × 80 mL). The combined dichloromethane
fractions were dried over MgSO4 and evaporated to give 0.96 g of
crude yellow oil. Subsequent purification of a 50-mg aliquot with
column chromatography (10% CH3OH in dichloromethane) af-
forded the product as a colorless oil (5 mg, 0.012 mmol, 8%, Rf )
0.05). 1H NMR (300 MHz, CDCl3) δ: 8.12 (d, 2H, benzyl-H), 7.99
(d, 2H, py-H), 7.78 (t, 2H, py-H), 7.54 (dd, 2H, py-H), 7.35 (d,
benzyl-H), 4.06-4.03 (m, 4H, py-CH2-NH), 3.97 (s, 6H, CH3),
3.04-2.60 (m, 5H, CH2-CH-CH2). 13C NMR (75 MHz, CDCl3)
δ: 165.9, 160.8, 147.7, 147.6, 147.4, 146.8, 137.7, 130.4, 125.9,
123.8, 58.5, 53.1, 52.6, 52.1, 39.6. HR-ESI-MS calcd. for
C25H28N5O6: 494.2040; found 494.2049 [M + H+]+.
(1,2-[N,N′-{p-Nitrobenzyl}methyl]-N,N′-[6-{methoxycarbonyl}-
pyridin-2-yl]methylamino)ethane (2). 1 was synthesized according
to the literature.17 4-Nitrobenzyl bromide (135 mg, 0.625 mmol)
was dissolved in 20 mL of acetonitrile together with 1 (105 mg,
0.293 mmol). Na2CO3 (400 mg) was added into the solution, and
the reaction was stirred overnight at 70 °C. Subsequently, the
suspension was filtered and the solvent removed in Vacuo. The
resulting orange oil was purified by column chromatography (Silica,
CH2Cl2); the product was eluted with 5% CH3OH and isolated as
an orange oil (60 mg, 0.095 mmol, 33%, Rf ) 0.6). 1H NMR (300
MHz, CDCl3) δ: 8.01 (d, 2H, benzyl-H), 7.98 (d, 2H, py-H), 7.76
(t, 2H, py-H), 7.59 (d, 2H, py-H), 7.46 (d, 2H, benzyl-H), 4.98 (s,
6H, CH3) 3.83 (s, 4H, py-CH2-NH), 3.69 (s, 4H, benzyl-CH2-NH),
2.71 (s, 4H, -CH2-CH2-). 13C NMR: (75 MHz, CDCl3) δ: 165.8,
160.1, 147.6, 147.3, 137.6, 129.3, 125.9, 123.8, 123.7, 60.6, 58.7,
53.1, 52.3. HR-ESI-MS calcd for C32H32N6NaO8: 651.2179; found:
651.2289 [M + Na]+.
(1,2-[N,N′-{p-Nitrobenzyl}methyl]-N,N′-bis-[6-carboxy-2-pyridyl-
methyl]ethylenediamine (3). 1 (27 mg, 0.042 mmol) was dissolved
in 4 mL of a 3:1 mixture of THF and water. LiOH (5 mg, 0.21
mmol) was added to the solution, resulting in an immediate color
change of the solution. The reaction was monitored by TLC and
found to be complete after 45 min. The solvent was removed in
Vacuo to afford a white solid (25 mg, 0.041 mmol, 97%). 1H NMR
(400 MHz, MeOD) δ: 8.12 (d, 2H, benzyl-H), 8.10 (d, 2H, py-H),
7.91 (t, 2H, py-H), 7.42 (d, 2H, py-H), 7.36 (d, 2H, benzyl-H),
3.88 (s, 4H, py-CH2-NH), 3.56 (s, 4H, benzyl-CH2-NH), 2.41 (s,
4H, -CH2-CH2-). 13C NMR (100 MHz, MeOD) δ: 172.5, 159.2,
155.0, 148.78, 145.0, 139.9, 132.2, 125.9, 124.4, 123.4, 61.0, 57.6,
31.1. HR-ESI-MS calcd for C30H27N6O8: 599.1890; found: 599.1887
[M - H+]-.
2-(p-Nitrobenzyl)-(1,2-[N,N′-{p-nitrobenzyl}methyl]-N,N′-bis-[6-
carboxy-2-pyridylmethyl]ethylenediamine (7). 6 (5 mg, 0.012
mmol) was dissolved in 2 mL of a 3:1 mixture of THF and water.
LiOH (1 mg, 0.04 mmol) was added into the solution. The reaction
was monitored by TLC and found to be complete after 30 min.
The solvent was removed in Vacuo to afford a light-yellow solid
1
(4 mg, 0.01 mmol, 83%). H NMR (400 MHz, CD3OD) δ: 8.15
(d, 2H, benzyl-H), 7.99 (d, 2H, py-H), 7.88 (t, 2H, py-H), 7.41 (m,
4H, py-H/benzyl-H), 4.12-3.91 (m, 4H, py-CH2-NH), 2.75-2.17
(m, 5H, CH2-CH-CH2). 13C NMR (100 MHz, CD3OD) δ: 165.0,
160.2, 155.2, 149.0, 148.1, 139.5, 131.6, 125.6, 124.6, 123.4, 59.5,
51.9, 39.9, 39.7. HR-ESI-MS calcd. for C23H22N5O6: 464.1570;
found: 464.1581 [M - H+].
[Ga(7)](NO3). 7 (2.5 mg, 5.3 µmol) was dissolved in water.
Ga(NO3)3 · 6H2O (2 mg, 5.5 µmol) was added, and the pH was
adjusted to 5 by addition of 0.1 M NaOH. The reaction mixture
was stirred at 60 °C for 2 h. The solvent was removed in Vacuo to
afford an off-white solid in quantitative yield. 1H NMR (300 MHz,
CD3OD) δ: 8.62 (t, 2H, py-H), 8.36 (d, 2H, benzyl-H), 8.20-8.09
(m, 4H, py-H), 7.53 (d, 2H, benzyl-H), 4.81-4.39 (m, 4H, py-
CH2-NH), 3.59-2.21 (m, 5H, CH2-CH-CH2). 13C NMR (150 MHz,
CD3OD) δ: 165.5, 165.4, 152.1, 151.6, 148.8, 147.4, 147.3 146.1,
145.9, 145.3, 131.8, 129.1, 129.0, 125.1, 124.7, 124.6, 58.9, 53.7,
51.5, 37.6. HR-ESI-MS calcd for C23H2169GaN5O6: 532.0748.;
found: 532.0743 [M]+.
[Ga(3)](NO3). 3 (7 mg, 0.011 mmol) was dissolved in a
CH3OH-water mixture (1:2). Ga(NO3)3 ·6H2O (4 mg, 0.011 mmol)
was added, and the pH was adjusted to 4.5 by addition of 0.1 M
NaOH. The reaction mixture was stirred at 60 °C for 2 h. The
solvent was removed in Vacuo to afford a white solid in quantitative
yield. The solid was redissolved in a mixture of water and methanol
(1:2). Colorless plates suitable for X-ray diffraction were obtained
[
67/68Ga(7)]+. 67GaCl3 or 68Ga3+ (100 µL, 1 mCi) in a 0.1 M
HCl solution was added into 10-4 M solution of 7 in 10 mM NaOAc
1
by slow evaporation of the solvent mixture. H NMR (400 MHz,
CD3OD) δ: 8.71 (t, 2H, py-H), 8.49 (d, 2H, py-H), 8.32 (d, 2H,
(18) Ali, M. S.; Quadri, S. Y. Bioconjugate Chem. 1996, 7, 576–583.
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15728 J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010

67/68Ga Complexes of Acyclic Chelates
A R T I C L E S
solution (pH 4) and left to react for 10 min at room temperature.
The reaction was monitored by analytical HPLC which showed
that the reaction had proceeded to 98%. Product tr on HPLC: 7.7
min (gradient: A - NaOAc buffer, pH 4.5; B - CH3OH, 0-100%
B linear gradient 20 min). For the apo-transferrin competition,
67GaCl3 was added to 10-4 M solution of 7 in 10 mM NaOAc
solution (pH 4.5). Complex formation was checked on HPLC
(peptide column). A 400-µL aliquot was added to a 1 mg/mL apo-
transferrin NaHCO3 solution (10 mM, 600 µL) and incubated at
37 °C (water bath). Complex stability was checked at time points
10 min, 1 h, and 2 h via analytical HPLC. The complex was 97%
intact after 2 h.
Solution Thermodynamics. Carbonate-free solutions of the
titrant, NaOH, were prepared by dilution of 50% solution (Acros
Organics) with freshly boiled MQ water under a stream of purified
nitrogen gas. The solution was standardized with potassium acid
phthalate, and the extent of carbonate accumulation was periodically
checked by titration with a standard hydrochloric acid solution and
determination of the corresponding Gran titration plot.19 Gallium
ion solutions were prepared by dilution of the appropriate atomic
absorption (AA) standard. The exact amount of acid present in the
gallium standard was determined by titration of an equimolar
solution of Ga and Na2H2EDTA. The amount of acid present was
determined by Gran’s method.19
Potentiometric titrations were performed using a Metrohm
Titrando 809 equipped with a Ross combination pH electrode and
a Metrohm Dosino 800. Data were collected in triplicate using PC
Control (Version 6.0.91, Metrohm). The titration apparatus consisted
of a 10 mL water-jacketed glass vessel maintained at 25.0 ( 0.1
°C (Julabo water bath). Prior to and during the course of the
titration, a blanket of nitrogen, passed through 10% NaOH to
exclude any CO2, was maintained over the sample solution. The
ionic strength was maintained at 0.16 M using NaCl. Prior to each
potentiometric equilibrium study, the electrode was calibrated using
standard HCl solutions. Calibration data were analyzed by standard
computer treatment provided within the program MacCalib20 to
obtain the calibration parameters E0 and pKw.
As the degree of formation of Ga(III) complexes at low pH (<2)
was too high for the determination of stability constants by use of
direct potentiometry, the ligand-ligand competition method using
Na2H2EDTA was also performed. Equilibrium was rapidly estab-
lished (less than 10 min); however, up to 15 min was permitted
between each titration point. The four successive proton dissociation
constants corresponding to hydrolysis of Ga(III) aqueous ion
included in the calculations were taken from Baes and Mesmer.21
The protonation constants of H2dedpa and stability constants of
Ga(III) were calculated from the experimental data using Hyperquad
2008.
X-ray Crystallography. Data for compound [Ga(dedpa)][ClO4]
were collected with graphite-monochromated Mo KR radiation
(0.71073 Å) at -17 °C on a Bruker X8 APEX II diffractometer.
The structure was solved using direct methods using SIR-9722 and
refined using SHELXL-97.23 All non-hydrogen atoms were refined
anisotropically. All N-H hydrogen atoms were located in a
difference map and refined isotropically. All other hydrogen atoms
were placed in calculated positions and refined using a riding model.
Data for compound [Ga(7)]ClO4 were collected with graphite-
monochromated Mo KR radiation at -183 °C on a Bruker APEX
DUO diffractometer. The structure was solved using direct methods
using SIR-9722 and refined using SHELXL-97.23 The material
crystallizes with two crystallographically independent moieties in
the asymmetric unit. One perchlorate anion is disordered and was
modeled in two orientations, with restraints used to maintain
reasonable geometries. Finally, MeOH solvent was found in the
lattice. Two molecules of solvent were located and modeled;
however, one region within the asymmetric unit had residual
electron density that could not be properly modeled. The
SQUEEZE24 program was used to generate a data set free of
residual electron density in that region. All non-hydrogen atoms
were refined anisotropically. All hydrogen atoms were placed in
calculated positions and refined using a riding model. A summary
of the relevant crystallographic data for both compounds can be
found in the Supporting Information.
Results and Discussion
In this contribution we report for the first time our study of
a linear platform chelate that has optimal properties in its
chelation of gallium. The use and the synthesis of H2dedpa
(originally25 named H2bpce) has been previously reported with
divalent metals showing reasonable chelation properties.25 In
the 1990s, our research group expended significant effort
investigating tripodal, nonmacrocyclic ligands containing amines,
pyridines, and carboxylates showing moderate chelation proper-
ties for Ga(III),26 but dedpa2- exceeds them all in its properties
of fast chelation with high specific activity at room temperature.
Most recently, we have synthesized a library of linear
hexadentate chelate ligands, and screened them for their ability
to bind 67Ga (a longer-lived Ga isotope that can serve as a model
for 68Ga). Under mild reaction conditions (room temperature,
aqueous buffer, pH 4), H2dedpa coordinated 67Ga quantitatively
within 10 min (as does NOTA27). DOTA however, requires
heating for quantitative reaction yields.28 Concentration-de-
pendent coordination of H2dedpa to both 68Ga and 67Ga at
concentrations as low as 10-7 M showed quantitative conversion
to the desired product. When coordinating to 68Ga, high specific
activities (as high as 9.8 ( 0.1 mCi nmol-1) were obtainable in
99% radiochemical yield without any purification steps. This
is the highest specific activity measured for any chelator with
68Ga, when neither heating nor 68Ga prepurification is used.29
Biodistribution Data. The protocol used in the animal studies
was approved by the Institutional Animal Care Committee of the
University of British Columbia and was performed in accordance
with the Canadian Council on Animal Care Guidelines. A total of
16 female ICR (20-30 g) mice were used for the animal study of
each of the three compounds. [67Ga(dedpa)]+, [67Ga(3)]+, or
[67Ga(7)]+ was prepared as described above and then diluted in
phosphate-buffered saline to a concentration of 100 µCi/mL. Each
mouse was i.v. injected with ∼10 µCi (100 µL) of the 67Ga complex
and then sacrificed by CO2 inhalation at 30 min, 1 h, 2 h, or 4 h
after injection (n ) 4 at each time point). Blood was collected by
cardiac puncture, and plasma was separated from whole blood by
centrifuging (2500 rpm, 15 min). Urine was collected from the
bladder. Tissues collected included kidney, liver, spleen, femur,
muscle, heart, lung, intestine, and brain. Tissues were weighed and
counted on a gamma counter, and the counts were converted to %
injected dose/gram (%ID/g).
(22) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giaco-
vazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna,
R. J. Appl. Crystallogr. 1999, 32 (1), 115–119.
(23) Sheldrick, G. M. SHELXL97-2: Program for the Refinement of Crystal
Structures, University of Go¨ttingen: Go¨ttingen, Germany, 1998.
(24) van der Sluis, P.; Spek, A. L. Acta Crystallogr., Sect. A 1990, 46,
194–201.
(25) Ferreiros-Martinez, R.; Esteban-Gomez, D.; Platas-Igesias, C.; de Blas,
A.; Rodriguez-Blas, T. Dalton Trans. 2008, 5754–5758.
(26) Caravan, P.; Rettig, C.; Orvig, C. Inorg. Chem. 1997, 36, 1306–1315.
(27) Velikyan, I.; Maecke, H.; Langstrom, B. Bioconjugate Chem. 2008,
19, 569–573.
(28) Meyer, G. J.; Maecke, H. R.; Schuhmacher, J.; Knapp, W. H.;
Hofmann, M. Eur. J. Nucl. Med. Mol. Imaging 2004, 31, 1097–1104.
(29) Velikyan, I.; Beyer, G. J.; Langstrom, B. Bioconjugate Chem. 2004,
15, 554–560.
(19) Gran, G. Analyst 1952, 77, 661–671.
(20) Duckworth, P. Private communication.
(21) Baes, C. F.; Mesmer, R. E. Wiley-Interscience: New York, 1976.
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A R T I C L E S
Boros et al.
relevant indicator of the extent to which a metal complex is
formed in solution is given by pM (-log[free M]) which
considers the influence of ligand basicity and chelate hydrolysis.
The values of log KML and pM of the Ga(III) complexes of
dedpa2- and other relevant multidentate ligands are shown in
Table 1. The high values of log KML and pM for [Ga(dedpa)]+
confirm the high affinity of dedpa2- for Ga(III) as well as high
thermodynamic stability.
The solid-state X-ray crystal structure (Figure 2) provides
significant insight into the coordination environment of
[Ga(dedpa)]+. In comparison with the crystallized Ga complexes
of NOTA11 and DOTA34 which have widely dispersed metal-
to-ligand bond distances, [Ga(dedpa)]+ has a more equally
distributed array of bond lengths, suggesting that the unusually
high stability of the complex is due to a near-perfect fit with
the Ga3+ ion (for further information about structural data see
the Supporting Information). Another characteristic that clearly
differentiates [Ga(dedpa)]+ from complexes with the macrocy-
clic chelators is the C2 rotational axis, which has also been
confirmed in solution through 13C NMR spectroscopy.
Figure 1. HPLC traces of [67Ga(dedpa)]+ Versus apo-transferrin in
competition; the trace of 67Ga-transferrin is shown for reference (gradient:
A - NaOAc buffer, pH 4.5; B - MeOH, 0-100% B linear gradient 20 min).
Table 1. Formation Constants (log KML) and pMa of Ga(III)
Complexes
ligand
log KML
pM
dedpa2-
28.11(8)
21.7
21.33
30.98
20.3
27.4
18.3
18.5
27.9
21.3
To investigate different modes of functionalization analogous
to the bifunctional versions of the macrocyclic chelators, two
model compounds 3 and 7 have been synthesized (Scheme 2).
Compound 3 displays derivatization through the two aliphatic
nitrogens, affording a scaffold capable of carrying two targeting
molecules, while compound 7 is derivatized through the
backbone of the ethylenediamine (en) component of the basic
ligand structure, retaining the original coordination environment
more closely, but only capable of carrying one targeting
molecule. Both 3 and 7 incorporate the nitrobenzyl functionality
which can be converted easily into the corresponding amino-
or isothiocyanato-benzyl, coupling moieties frequently employed
for conjugation to target molecules via a free carboxylate or
primary amine, respectively.35,36
EDTA31
DOTA32
NOTA33
transferrinb
a Calculated for 10 µM total ligand and 1 µM total metal at pH 7.4
and 25 °C. b Conditional constant for log KML from ref 30.
To investigate the stability of the 67Ga radiochemical complex,
a 2-h competition experiment was conducted in the presence
of excess human apo-transferrin; the iron-sequestering/-transport
protein that has very high affinity for Ga(III) (Figure 1).30 The
[67Ga(dedpa)]+ complex was fully intact after 2 h, suggesting
that it should have very high in ViVo stability, similar to that
reported for 68Ga NOTA complexes.16 In a direct competition
for chelation of 67Ga with equal concentrations of both NOTA
and H2dedpa, over 96% was coordinated by (dedpa)2-, less than
1% by NOTA, demonstrating the expected faster Ga complex-
ation with the acyclic H2dedpa than with macrocyclic NOTA.
Solution thermodynamic investigations of the corresponding
cold complex [Ga(dedpa)]+ have provided a complex stability
constant of log KML ) 28.11(8), obtained by ligand-ligand
competition with EDTA using potentiometric titration. A more
Compound 3 is synthesized from 1,2-[{6-(methoxycarbon-
yl)pyridin-2-yl}methylamino]ethane,17 which is then subse-
quently alkylated with 4-nitrobenzyl bromide (see the Supporting
Information). Intermediate 2 is purified and the carboxylates
are deprotected under standard conditions to afford the clean
product 3 as a white solid. The complex with cold (nonradioac-
tive) gallium(III) is formed within 2 h at pH 4-5 under gentle
heating (Figure 3), and again the C2 rotational axis was con-
Figure 2. Solid-state structure of the cation in [Ga(dedpa)][ClO4]; relevant bond lengths [Å]: N1-Ga: 1.9866(16); N2-Ga: 1.9902(16); N3-Ga: 2.1115(16);
N4-Ga: 2.1132(16); O1-Ga: 1.9708(13); O2-Ga: 1.9828(13).
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67/68Ga Complexes of Acyclic Chelates
A R T I C L E S
Scheme 2. Syntheses of Compounds 3 and 7a
a Reagents and conditions: a) 4-nitrobenzyl bromine, Na2CO3, CH3CN, 18 h; b) LiOH, THF/water (3:1), 45 min; c) CH3OH, reflux, 2 h; d) NaBH4, 0°C,
2 h; e) LiOH, THF/water (3:1), 30 min.
Table 2. Stability of Investigated Chelators at Various Times in the
2 h Competition Experiment in the Presence of Excess Human
apo-Transferrin
ligand
10 min (%)
1 h (%)
2 h (%)
H2dedpa
3
7
>99
88
98
>99
69
97
>99
51
97
Compound 7 is furnished through a different route; 4 is
derived from 4-nitro-L-phenylalanine,18 while 5 is afforded
through a four-step synthesis from 2,6-pyridinedicarboxylic
acid.17 A one-pot reductive amination process produces 6 in
moderate yields along with a mixture of impurities, which can
be separated from the product through column chromatography.
The subsequent deprotection leads to 7 as a light-orange solid.
The complex with cold gallium(III) is formed within 2 h at pH
4-5 under gentle heating, whereas the 67Ga and 68Ga complexes
are formed within 10 min at room temperature in 97%
radiochemical yield. The subsequent apo-transferrin challenge
experiment reveals a stability comparable to that of [Ga(d-
edpa)]+, with over 97% of the complex remaining intact after
2 h (traces, see Supporting Information). Concentration-depend-
ent coordination to 68Ga showed that 7 is capable of coordinating
under standard, mild conditions at concentrations as low as 10-6
M much like 3.
The biodistribution study in mice (Figure 4, raw data in the
Supporting Information) indicated that [67Ga(dedpa)]+ cleared
from the background tissue, such as muscle, within the first 30
min and was excreted mainly through the kidneys. The in ViVo
stability of [67Ga(dedpa)]+ was supported by the low uptake in
bone, which is known to be a site of increasing accumulation
for weakly chelated 67Ga.37 The overall biodistribution profile
compares well to that of macrocyclic chelators evaluated in a
Figure 3. Solid state structure of the cation in [Ga(3)][ClO4]; relevant bond
lengths [Å]: N1-Ga: 1.992(5); N2-Ga: 1.981(5); N3-Ga: 2.188(5);
N4-Ga: 2.159(5); O1-Ga: 1.967(4); O2-Ga: 1.976(4).
firmed in both the solid-state structure and the solution NMR
spectrum (see the Supporting Information). Coordination to 67Ga
or 68Ga forms the complex within 10 min at room temperature
in 98% radiochemical yield. The subsequent apo-transferrin
challenge experiment revealed 51% of the radiolabeled complex
remained intact after 2 h in the presence of excess apo-
transferrin; a stability inferior to that of [Ga(dedpa)]+, but
comparable to that of Ga-DOTA (for data see the Supporting
Information).16 Concentration-dependent coordination to 68Ga
showed that 3 is capable of coordinating under standard, mild
conditions at concentrations as low as 10-6 M (Table 2).
(30) Harris, W. R.; Pecoraro, V. L. Biochemistry 1983, 22, 292–299.
(31) Bottari, E.; Anderegg, G. HelV. Chim. Acta 1967, 50, 2349–2356.
(32) Clarke, E. T.; Martell, A. E. Inorg. Chim. Acta 1991, 190, 37–46.
(33) Clarke, E. T.; Martell, A. E. Inorg. Chim. Acta 1991, 181, 273–280.
(34) Heppeler, A.; Froidevaux, S.; Maecke, H. R.; Jermann, E.; Be´he´, M.;
Powell, P.; Hennig, M. Chem.sEur. J. 1999, 5, 1016–1023.
(35) McMurry, T. J.; Brechbiel, M.; Kumar, K.; Gansow, O. A. Biocon-
jugate Chem. 1992, 3, 108–117.
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A R T I C L E S
Boros et al.
collected as an additional data point (Figure 5, raw data in the
Supporting Information). Both [67Ga(3)]+ and [67Ga(7)]+ exhibit
improved clearance from all organs, low bone uptake (indicator
for complex stability) and excretion through urine. Despite lower
in Vitro stability, the biodistribution of [67Ga(3)]+ suggests high
stability in ViVo and shows better clearance from blood and
kidneys than both [67Ga(7)]+ and [67Ga(dedpa)]+. It is possible
that compounds containing secondary amines associate stronger
with blood serum proteins and kidney tissue; however, the
difference in biodistribution of even [67Ga(7)]+ and [67Ga(d-
edpa)]+ shows that added functionalities, such as peptides or
other targeting vectors, have a great impact on the interaction
of these compounds with in ViVo systems.
Figure 4. Biodistribution over 4 h of [67Ga(dedpa)]+ in female ICR mice.
Conclusion
similar study,16 also exhibiting the low uptake in liver and
intestines that is characteristic of ionic compounds. The per-
sistent high uptake in the blood serum was not confirmed with
the derivatized compounds, suggesting that added functionality
influences biodistribution. Serum stability studies done in Vitro
confirmed that [67Ga(dedpa)]+ was stable to transchelation by
serum proteins.
H2dedpa complexes quickly with Ga and forms complexes
of very high stability, comparing well to the widely used
macrocyclic chelator NOTA and exceeding the properties of
DOTA. H2dedpa and its derivatives can be coordinated to Ga
isotopes under mild room-temperature conditions at high specific
activities in short reaction times, making it an ideal scaffold
for further elaboration and applications such as peptide labeling.
The high radiochemical yield and high specific activity of the
products could obviate the need for time-consuming HPLC
purification, a major advantage for the short-lived isotope, 68Ga.
In addition, the biodistributions of [67Ga(dedpa)]+, [67Ga(3)]+,
In the biodistribution studies for [67Ga(3)]+ and [67Ga(7)]+,
whole blood was collected instead of serum, and urine was
(36) Wei, L.; Ye, Y.; Wadas, T. J.; Lewis, J. S.; Welch, M. J.; Achilefu,
S.; Anderson, C. J. Nucl. Med. Biol. 2009, 36, 277–285.
Figure 5. Biodistribution of [67Ga(3)]+ (above) and [67Ga(7)]+ (below) in female ICR mice over 4 h; complete data for urine is also shown in separate
diagrams to the right.
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67/68Ga Complexes of Acyclic Chelates
A R T I C L E S
and [67Ga(7)]+ confirm the stability of the complexes measured
in Vitro with general clearance, rendering these frameworks a
good basis for elaboration of new Ga bioconjugates.
It is important to note that many of the advantageous
properties described have been observed previously with only
one macrocyclic chelate (NOTA), while they are unexpected
for an acyclic system. We are currently applying these results
to small biomolecules and other radiometals to evaluate the true
potential of this exciting chelation scaffold.
and Engineering Research Council (NSERC) of Canada for grant
support and a CGSM fellowship (E.W. P.), and Dr. Dawn Water-
house of the BC Cancer Agency for carrying out the biodistribution
study.
Supporting Information Available: Detailed information on
potentiometric measurements, X-ray structure determinations
(including tables of bond lengths and angles), detailed HPLC
radio-traces, 1H NMR spectra of key compounds, and detailed
data of all biodistribution studies. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We acknowledge UBC for a Four Year
Fellowship (E.B.), MDS Nordion (Canada) and the Natural Sciences
(37) Sephton, R. G.; Hodgson, G. S.; De Abrew, S.; Harris, A. W. J. Nucl.
Med. 1978, 19 (8), 930–935.
JA106399H
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J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010 15733