High-denticity ligands based on picolinic acid for 111In radiochemistry

Article abstract of DOI:10.1139/cjc-2013-0542

Four new acyclic ligands, Bn-H₃nonapa (3), H₃nonapa (4), p-NO₂-Bn-H₃nonapa (10), and Bn-H₃trenpa (7), were synthesized and studied with nonradioactive In³⁺ and with radioactive ¹¹¹

Full text of DOI:10.1139/cjc-2013-0542

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1
ARTICLE
High-denticity ligands based on picolinic acid for 111In
radiochemistry
Eric W. Price, Cara L. Ferreira, Michael J. Adam, and Chris Orvig
Abstract: Four new acyclic ligands, Bn-H3nonapa (3), H3nonapa (4), p-NO2-Bn-H3nonapa (10), and Bn-H3trenpa (7), were synthe-
sized and studied with nonradioactive In3+ and with radioactive 111In3+. The coordination of these ligands to In3+ was confirmed
by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Radiolabeling experiments were per-
formed with 111In3+; these demonstrated H3nonapa (4) to be the best indium ligand of those studied herein, achieving radio-
chemical yields of ϳ97% in 10 min at ambient temperature, and stability to transchelation in mouse serum of 44.5% 25.9% after
24 h. Although the radiolabeling kinetics of H3nonapa (4) were excellent, serum stability results were inferior to the previously
studied ligands DOTA, DTPA, and H4octapa, suggesting that the presented ligands may find their optimum radiopharmaceutical
applications with isotopes other than 111In. Owing to the high denticity of these ligands (9–10 coordinate), they may realize their
potential with large ion isotopes such as 177Lu, 86/90Y, and 225Ac.
Key words: radiopharmaceuticals, coordination chemistry, radiometals, multidentate ligands, picolinates.
Résumé : Quatre nouveaux ligands acycliques Bn-H3nonapa (3), H3nonapa (4), p-NO2-Bn-H3nonapa (10) et Bn-H3trenpa (7) ont été
synthétisés et étudiés a` l'aide de l'ion In3+, non radioactif, et de l'ion 111In3+, radioactif. La coordination de ces ligands avec l'ion
In3+ a été confirmée par spectrométrie de masse de haute résolution et spectroscopie de résonance magnétique nucléaire. Des
expériences de radiomarquage avec l'ion 111In3+ ont été réalisées et ont montré que H3nonapa (4) était le plus performant des
quatre ligands étudiés, de par son rendement chimique (environ 97%, après 10 minutes et a` la température ambiante) et sa
stabilité a` l'égard de la transchélation dans du sérum de souris (44,5% 25,9% des ligands intacts après 24 h). Bien que la cinétique
du radiomarquage de H3nonapa (4) fût excellente, les résultats de la stabilité de ce dernier dans le sérum ont été inférieurs a` ceux
obtenus pour les ligands précédemment étudiés (DOTA, DTPA et H4octapa), ce qui signifie que les ligands étudiés ici pourraient
être utilisés de façon optimale dans le domaine pharmaceutique avec des isotopes autres que l'indium 111In. En raison de la
denticité élevée de ces ligands (coordinence de 9 a` 10), ils pourraient atteindre tout leur potentiel avec des isotopes ioniques
lourds tels que 177Lu, 86/90Y et 225Ac. [Traduit par la Rédaction]
Mots-clés : radiopharmaceutiques, chimie de coordination, radiométaux, ligands multidentates, picolinates.
chelator diethylenetriaminepentaacetic acid),8,16–19 and the
recent discovery H4octapa (N,N-bis(6-carboxyl-2-pyridylmethyl)-
Introduction
The radiometal 111In is a versatile isotope that has been com-
monly used for single photon emission computed tomography
(SPECT) and Auger electron/␤– therapy, typically withtargeting
vectors such as peptides, antibodies, and nanoparticles.1–6 Imag-
ing agents based on 111In are often used for pretherapy scouting
scans to obtain dosimetry information, which is then followed by
injection of the same agent prepared with a therapeutic isotope
such as 177Lu (half-life (t1/2) ϳ 6.6 d, ␤– therapy) or 90Y (t1/2 ϳ 2.7 d,
␤– therapy).1–6 The fundamental core of any radiometal-based ra-
diopharmaceutical is the ligand, which serves as an anchor for
chelating and sequestering radiometals, and must be chosen so
that it forms extremely stable complexes that resist transchela-
tion and demetallation in vivo.
ethylenediamene-N,N-diacetic acid).20–24 A newly published acy-
clic bipyridine-chelator (BPCA) has also shown promise for fast
ambient temperature radiolabeling of 111In and strong in vivo
stability.3,25,26 Other than H4octapa and BPCA, not many new li-
gands have been published for use with 111In in recent years, with
most efforts being focused on newer positron emission computed
tomography (PET) isotopes like 68Ga.1–6,27 The macrocyclic ligand
DOTA (Chart 1) has been the main workhorse for radiochemical
research with the radiometals 111In, 177Lu, and 86/90Y. Macrocycles
have traditionally been favored over acyclic ligands because they
are generally more kinetically inert than acyclic chelators, largely
as a result of their constrained geometries and partially preorga-
nized binding cavities.8,9,28–33 The major drawbacks with macro-
cycles such as DOTA are the requisite heating (40–90 °C) and
extended reaction times (30–180 min) needed for quantitative
There are a number of ligands that perform adequately for
radiolabeling with 111In, such as DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid),7–16 CHX-A== -DTPA (derivative of acyclic
Received 21 November 2013. Accepted 17 January 2014.
E.W. Price. Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1,
Canada; TRIUMF, 4004 Wesbrook Mall, Vancouver, BC V6T 2A3, Canada.
C.L. Ferreira. Nordion, 4004 Wesbrook Mall, Vancouver, BC V6T 2A3, Canada.
M.J. Adam. TRIUMF, 4004 Wesbrook Mall, Vancouver, BC V6T 2A3, Canada.
C. Orvig. Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1,
Canada.
Corresponding authors: Michael J. Adam (e-mail: adam@triumf.ca) and Chris Orvig (e-mail: orvig@chem.ubc.ca).
This article is part of a Special Issue dedicated to Professor Barry Lever in recognition of his contributions to inorganic chemistry across Canada and beyond.
Dedicated, with the greatest respect, affection, and admiration, to Barry Lever, guru to C.O.
Can. J. Chem. 92: 1–11 (2014) dx.doi.org/10.1139/cjc-2013-0542
Published at www.nrcresearchpress.com/cjc on 22 January 2014.

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Can. J. Chem. Vol. 92, 2014
Chart 1. The previously studied chelating ligands H2dedpa, H4octapa, H5decapa, DOTA, and DTPA, and the new compounds Bn-H3nonapa,
H3nonapa, p-NO2-Bn-H3nonapa, and Bn-H3trenpa.
radiolabeling; these conditions are unsuitable for sensitive vec-
Results and discussion
The four novel ligands Bn-H3nonapa (3), H3nonapa (4), p-NO2-
Bn-H3nonapa (10), and Bn-H3trenpa (7) were synthesized using the
tors such as antibodies.8,9,28–40 The acyclic chelator diethyl-
enetriaminepentaacetic acid (DTPA) (as with most acyclic ligands)
exhibits much faster reaction kinetics than DOTA and can radio-
same general methods used for the original synthesis of the ligand
H2depda.23,44–46 The amine backbones diethylenetriamine (dien)
and tris(2-aminoethyl)amine (tren) were chosen as platforms for
assembling high-denticity derivatives of H2dedpa and H4octapa
(N,N–bis(6-carboxyl-2-pyridylmethyl)ethylenediamine), although
any permutation of these backbones could be theoretically used.
Dien and tren were benzyl-protected via reductive amination with
benzaldehyde to allow for controlled alkylation of bromopicolinic
acid in subsequent steps (Schemes 1, 2, and 3). After alkylation of
bromopicolinic acid, benzyl groups could be removed via hy-
drogenation in glacial acetic acid, or benzyl groups could be re-
tained by bypassing this step (Schemes 1–3). For the ligand
H3nonapa (4, Scheme 1), hydrogenation was performed to remove
benzyl-protecting groups, followed by deprotection by refluxing in
HCl (12 mol/L) or stirring with LiOH at ambient temperature. Benzyl
groups were used as placeholders for p-NO2-benzyl groups (e.g.,
p-NO2-Bn-H3nonapa (10), Scheme 3), which have the potential to be
transformed into p-SCN-benzyl groups to yield active bifunctional
derivatives. Benzyl-isothiocyanate groups are commonly used for
facile conjugation reactions to free primary amine groups (e.g., anti-
bodies and peptides), forming stable thiourea bioconjugates. Al-
though excess benzyl groups can result in undesirably high
lipophilicity, this is only an issue for peptide conjugates, and will not
be an issue when conjugated to large targeting vectors like antibod-
ies and nanoparticles. The cumulative yield obtained for H3nonapa
(4) was ϳ24% in four steps, which was significantly lower than that
for Bn-H3nonapa, which was synthesized in a cumulative yield of
label quantitatively in a matter of minutes at ambient tempera-
ture, but the resulting complexes are not nearly as stable in vivo
as those of DOTA (Chart 1).30,31,41–43 New acyclic ligands with rapid
ambient temperature radiolabeling kinetics, robust in vivo stabil-
ity and inertness (compared to macrocycles such as DOTA), and a
variety of physical properties (e.g., charge, denticity, and donor
atoms) are especially important for antibody vectors; these highly
successful vectors are used for targeting a variety of cancers and
cannot withstand the elevated reaction temperatures ideally re-
quired for macrocycles like DOTA.30,31,41–43
In this work, we report the synthesis of four new acyclic ligands
based on picolinic acid. These ligands have denticities ranging
from 9 to 10, and are based on either a diethylenetriamine
(dien) or tris(2-aminoethyl)amine (tren) backbone. The ligands
Bn-H3nonapa (N,N== -[(benzyl)-N,N=,N== -[(6-carboxy)pyridine-2-yl]methyl]-
diethylenetriamine; 3), H3nonapa (N,N=,N== -[(6-carboxy)pyridine-2-yl)-
methyl]diethylenetriamine; 4), p-NO2-Bn-H3nonapa (N,N== -[(p-nitroben-
zyl)-N,N=,N== -[(6-carboxy)pyridine-2-yl]methyl]diethylenetriamine;
10), and Bn-H3trenpa (N,N=,N== -tris[benzyl]tris[(6-carboxy)pyridine-
2-yl)methyl]ethylamine; 7) have been synthesized and character-
ized for the first time (Chart 1). The nonradioactive In3+ complexes
of ligands 3, 4, and 10 were synthesized and characterized. These
ligands were also studied with the radiometal ion 111In3+, and
assessed by radiolabeling experiments and mouse serum stability
assays.
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Scheme 1. Synthesis of Bn-H3nonapa (3) and H3nonapa (4). (i) Diethylenetriamine (dien), benzaldehyde, MeOH, Ar (g), ⌬, 2 h; (ii) MeOH, 0 °C,
NaBH4, 16 h, 62%; (iii) methyl-6-bromomethylpicolinate, CH3CN, 60 °C, 20 h, 81%; (iv) tetrahydrofuran (THF)/H2O (3:1), LiOH, RT, 2 h,
Bn-H3nonapa (3) 93%; (v) AcOH, Pd/C (10 wt%), H2 (g), RT, 16 h; (vi) HCl (12 mol/L), ⌬, 16 h, H3nonapa (4) 47%.
Scheme 2. Synthesis of Bn-H3trenpa (7). (i) Tris(2-aminoethyl)amine (tren), benzaldehyde, MeOH, Ar (g), ⌬, 2 h; (ii) MeOH, 0 °C, NaBH4, 16 h,
17%; (iii) methyl-6-bromomethylpicolinate, CH3CN, 60 °C, 20 h, 64%; (iv) THF/H2O (3:1), LiOH, RT, 2 h, Bn-H3trenpa (7) 80%.
ϳ47% in three steps. This decreased yield is largely a result of a
problem previously reported for the synthesis of H4octapa, where it
was determined that hydrogenation conditions resulted in the cleav-
age of both benzyl and picolinic acid moieties, producing significant
quantities of byproduct and substantially decreasing yields and com-
plicating purification of final products.22 This inefficiency was
largely obviated in the synthesis of H4octapa by not performing the
hydrogenation reaction in the presence of the picolinic acid moiety;
however, for the synthesis of the currently discussed ligands this was
not possible.22
cursor (compound 6, Scheme 2). In addition to cleaving benzyl
and picolinic acid moieties from the tren backbone, it was also
observed by mass spectrometry that hydrogenation conditions
cleaved the ethylene bridges of tren. For this reason, the tren-
based ligand, H3trenpa, analogous to the dien-based ligand,
H3nonapa, could not be produced. Additionally, p-NO2-Bn-H3trenpa
could not be synthesized, as the target ligand was found to be
unstable and decomposed during synthesis. This instability was
also observed in the synthesis of p-NO2-Bn-H3nonapa (10), al-
though to a lesser degree (Scheme 3). In light of the synthetic
issues plaguing Bn-H3trenpa (7), it was determined to be the least
robust ligand of those presented in this work, and synthesis of a
p-NO2-Bn bearing a bifunctional precursor was attempted but
The synthesis of Bn-H3trenpa (7) was more challenging than
that of H3nonapa (4)/Bn-H3nonapa (3), as the hydrogenation reac-
tion was found to completely decompose the Bn-(Me)3trenpa pre-
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Can. J. Chem. Vol. 92, 2014
Scheme 3. Synthesis of p-NO2-Bn-H3nonapa (10). (i) Diethylenetriamine (dien), p-4-nitrobenzaldehyde, MeOH, Ar (g), ⌬, 2 h; (ii) MeOH, 0 °C,
NaBH4, 16 h, 40%; (iii) methyl-6-bromomethylpicolinate, CH3CN, 60 °C, 20 h, 64%; (iv) THF/H2O (3:1), LiOH, RT, 2 h, p-NO2-Bn-H3nonapa (10) 16%.
could not be completed, suggesting little potential of this tren-
based ligand system for use in 111In radiopharmaceuticals.
blood–brain barrier, and so investigation into their stability is still
warranted.48
Ligand 10 (p-NO2-Bn-H3nonapa) was synthesized in the same
manner as Bn-H3nonapa, with the only modification to the
procedure being the substitution of p-NO2-benzaldehyde for
benzaldehyde in the initial reductive amination reaction. Despite
the synthetic steps being identical, the p-NO2-Bn functionality
appeared to be more labile and sensitive to decomposition than
the regular benzyl groups, decreasing yields and complicating
purifications. Ligand 10 was produced in three synthetic steps
with a low cumulative yield of ϳ4%, demonstrating the poor sta-
bility of the p-NO2-Bn functionality because the analogous synthe-
sis of Bn-H3nonapa (3) was also completed in three steps, albeit in
yields of ϳ47%. The bifunctional precursor p-NO2-Bn-H3nonapa
(10) was not transformed into the active isothiocyanate, although
it can be expected that the requisite hydrogenation conditions
would effect further decomposition and decreased yields. A
synthetic methodology to circumvent the destructive effects of
hydrogenation reactions on this system was previously deter-
mined, where instead of the p-NO2-Bn groups, tert-butyl ester pro-
tected p-NH2-Bn functionality was used successfully.47 Should the
radiolabeling efficiency and blood serum stability of these ligands
with 111In have been promising enough (vide infra), this synthetic
revision could be applied to the presented ligands to more effi-
ciently produce bifunctional derivatives.47 If these ligands were to
The complex In(nonapa) (12) was the most soluble in water and
provided the highest radiochemical yields with 111In, and addi-
tionally demonstrated the highest stability in mouse serum (vide
infra). It can be observed that the NMR spectra of the free ligands
displayed sharp and well-resolved peaks, but upon coordination
to In3+, the 1H NMR spectra became much more complicated and
broad (Figs. 1–3). In(Bn-nonapa) (11) displayed the most significant
broadening of peaks, suggesting rapid fluxional isomerization in
solution (Fig. 2). The NMR spectrum of In(nonapa) (12, Fig. 1) be-
came much more complicated with many new signals being
observed compared to the free ligand, but the NMR peaks still
appeared sharp and well resolved, suggesting little fluxional
isomerization and a decrease in symmetry and (or) the presence of
multiple static isomers giving rise to a more complex spectrum.
In(Bn-trenpa) (13, Fig. 3) showed an NMR spectrum somewhere
between those of In(nonapa) (12) and In(Bn-nonapa) (11), display-
ing many new signals and broadening of signals to a lesser degree
than that observed for In(Bn-nonapa) (11). Metal–ligand com-
plexes that form highly symmetric complexes with no fluxional
isomerization typically show much more simple NMR spectra
than those observed here, as in the spectra obtained previously
for [Ga(dedpa)]+ and [In(octapa)]–.22,23 Although it is interesting to
obtain a general idea of the behavior and isomerization of these
complexes in solution, to properly assess their potential in vivo as
radiopharmaceuticals they must be studied using radiolabeling
and serum stability experiments.
86/
find purpose in the future with other radiometals (e.g., 177Lu,
90Y, or 225Ac), a combination of this tert-butyl ester protected
p-NH2-Bn functionality and the recently devised nosyl-protection
strategy should provide reasonable synthetic access to fully bi-
functional derivatives.20,21,47
The new acyclic ligands H3nonapa (4), Bn-H3nonapa (3), and
Bn-H3trenpa (7) were radiolabeled with 111In under conditions sim-
ilar to those in our previous work with the acyclic chelating ligand
H4octapa (NaOAc buffer, pH 4.5, room temperature (RT)).22 It was
found that the potentially nonadentate ligand H3nonapa effi-
ciently radiolabeled 111In, providing a radiochemical yield of ϳ97%
after 10 min at ambient temperature (Table 1, Fig. 4). The ben-
zylated derivative of H3nonapa (Bn-H3nonapa) achieved less opti-
mal radiolabeling kinetics, with a still respectable yield of ϳ93%
after 45 min at ambient temperature. The potentially decaden-
tate, tren-based ligand, Bn-H3trenpa, displayed poor radiolabeling
performance, with a radiochemical yield of only ϳ15% after
45 min at room temperature. Elevated temperatures may have
increased the radiochemical yield of Bn-H3trenpa beyond ϳ15%;
however, the purpose of these acyclic ligands is to provide facile
room temperature radiolabeling kinetics and so this eventuality
was not explored.
The ligands Bn-H3nonapa (3), H3nonapa (4), and Bn-H3trenpa (7)
were synthesized and obtained as their HCl salts, and were re-
acted with indium perchlorate (In(ClO4)3·6H2O) under aqueous
conditions (pH 4–5). The coordination complexes were confirmed
via high-resolution mass spectrometry (HR-MS) and nuclear mag-
netic resonance (NMR) spectroscopy. Coordination to In3+ was
confirmed by significant changes in chemical shifts and coupling
patterns in their NMR spectra (Figs. 1, 2, and 3). The NMR spectra of
the indium complexes were obtained in deuterated dimethyl
sulfoxide (DMSO-d6), as the solubility of these neutral indium
complexes was very poor in water and methanol. Because these
ligands are designed with in vivo radiopharmaceutical applica-
tions in mind, their partial insolubility in water is not ideal;
however, under the extremely dilute conditions used in radio-
chemistry, and when coupled to highly soluble targeting vectors
such as peptides and antibodies, such insolubility can be over-
come.47 Additionally, highly stable but lipophilic compounds can
sometimes be applied to cardiac perfusion imaging or passing the
Once radiolabeled with 111In, the radiometal complexes were
incubated with mouse blood serum as a transchelation challenge
(e.g., to transferrin in serum) to estimate their in vivo inertness.
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Price et al.
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Fig. 1. 1H NMR spectra of H3nonapa (4; 400 MHz, 25 °C, D2O, top) and the In3+ complex In(nonapa) (12; 600 MHz, 25 °C, DMSO-d6, bottom).
111In(nonapa) demonstrated excellent stability in mouse serum
after 1 h of 93.5% 0.4%, but after 24 h had undergone significant
transchelation with only 44.5% 25.9% remaining intact (Fig. 5). Of
the three new ligands investigated, H3nonapa was the most prom-
ising, with 111In(Bn-nonapa) remaining only 16.6% 0.4% intact
after 24 h in mouse serum, and 111In(Bn-trenpa) providing insuffi-
cient radiolabeling yields (ϳ15%) to warrant evaluation by a serum
stability challenge. It was observed that the serum stability and
radiolabeling efficiency were decreased for 111In complexes of Bn-
H3nonapa compared to that of H3nonapa (Table 1, Fig. 5), suggest-
ing that benzylating the secondary amines of H3nonapa to tertiary
amines with Bn-H3nonapa may have resulted in weaker coordina-
tion to indium, which was therefore more easily transchelated by
serum proteins. This is a trend that was observed for the gallium
complex of H2dedpa, where alkylating the secondary amines to
tertiary amines generally resulted in decreased stability and inert-
ness to transchelation in apo-transferrin and serum stability chal-
lenges.23,47,48 It is important to note that macroscale synthesis of
nonradioactive In(Bn-nonapa)] produced a water insoluble com-
plex, and although poor solubility is expected to be less of a prob-
lem at the extremely dilute conditions used for radiolabeling with
111In, it may have contributed to the inferior stability with 111In by
means of precipitation or nonspecific association with serum pro-
teins. In consideration of the superior radiolabeling and serum
stability properties of the previously studied chelators H4octapa,
H5decapa, DOTA, and DTPA, the three new ligands, H3nonapa (4),
Bn-H3nonapa (3), and Bn-H3trenpa (7), were not investigated for in
vivo applications with 111In.20,22 These new ligands may prove
useful in the future for larger radiometals that could benefit from
their high denticities, such as 177Lu, 86/90Y, and 225Ac.
Conclusions
The four new acyclic ligands Bn-H3nonapa (3), H3nonapa (4),
p-NO2-Bn-H3nonapa (10), and Bn-H3trenpa (7) were synthesized
and studied with nonradioactive In3+, and with radioactive 111In3+
.
The coordination of these ligands to In3+ was confirmed by high-
resolution mass spectrometry and nuclear magnetic resonance
1
spectrometry. The H NMR spectra of the complexes In(nonapa)
(12), In(Bn-nonapa) (11), and In(Bn-trenpa) (13) revealed broad
and complicated splitting patterns, suggesting low symmetry
and (or) fluxional isomerization, or the presence of multiple iso-
mers in solution. The poor solubility of the In(Bn-nonapa) and
In(Bn-trenpa) complexes in aqueous conditions was not promis-
ing for potential in vivo applications. Radiolabeling experiments
were performed with 111In3+, which demonstrated H3nonapa (4) to
be the most proficient ligand studied herein, achieving radio-
chemical yields of ϳ97% in 10 min at ambient temperature, and
possessing stability to transchelation by mouse serum of 44.5%
25.9% after 24 h. Although the radiolabeling kinetics of H3nonapa
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Can. J. Chem. Vol. 92, 2014
Fig. 2. 1H NMR spectra of Bn-H3nonapa (3; 300 MHz, 25 °C, MeOD, top) and the In3+ complex In(Bn-nonapa) (11; 600 MHz, 25 °C, DMSO-d6,
bottom).
(4) were excellent, serum stability results were inferior to the
previously studied ligands DOTA, DTPA, and H4octapa, suggesting
that these ligands are not robust enough for 111In radiopharma-
ceutical applications. The ligand Bn-H3nonapa (3) possessed
poor radiolabeling properties with 111In, requiring 45 min at
ambient temperature to achieve radiochemical yields of ϳ93%,
and demonstrating poor serum stability (16.6% 0.4% after 24 h).
The ligand Bn-H3trenpa (7) was only able to achieve radiochemical
yields of ϳ15% after 45 min at ambient temperature, which was
deemed unacceptable for further study. Owing to the high denticity
of these ligands (potentially 9–10 coordinate), they may have value
for study with large isotopes like 177Lu, 86/90Y, and 225Ac.
60 Å pore size, 40–63 mm particle size, Silicycle, Quebec, QC).
Automated column chromatography was performed using a Tele-
dyne Isco (Lincoln, NE) CombiFlash Rf automated system with
solid load cartridges packed with flash column silica gel and Re-
diSep Rf Gold reusable normal-phase silica columns and neutral
1
alumina columns (Teledyne Isco, Lincoln, NE). H and 13C NMR
spectra were recorded on Bruker AV300, AV400, or AV600 instru-
ments; all spectra were internally referenced to residual solvent
peaks except for 13C NMR spectra in D2O, which were externally
referenced to a sample of CH3OH/D2O. Low-resolution mass spec-
trometry was performed using a Waters liquid chromatography-
mass spectrometer (LC–MS) consisting of a Waters ZQ quadrupole
spectrometer equipped with an electrospray/chemical ionization
(ESCI) ion source and a Waters 2695 high-performance liquid
chromatography (HPLC) system (Waters, Milford, MA). High-
resolution electrospray-ionization mass spectrometry (HR-EI-MS)
was performed on a Waters Micromass LCT time of flight instru-
ment. Microanalyses for C, H, and N were performed on a Carlo
Erba EA 1108 elemental analyzer. The HPLC system used for puri-
fication of nonradioactive compounds consisted of a semiprepara-
tive reverse phase C18 Phenomenex synergi hydro-RP (80 Å pore
size, 250 mm × 21.2 mm, Phenomenex, Torrance, CA) column
connected to a Waters 600 controller, a Waters 2487 dual wave-
length absorbance detector, and a Waters delta 600 pump.
111In(chelate) mouse serum stability experiments were analyzed
Experimental
General materials and methods
All solvents and reagents were purchased from commercial sup-
pliers (Sigma-Aldrich, St. Louis, MO; TCI America, Portland, OR;
Fisher Scientific, Waltham, MA) and were used as received unless
otherwise indicated. Methyl-6-bromomethylpicolinate was syn-
thesized according to a literature protocol.22 Water used was ul-
trapure (18.2 M⍀ cm−1 at 25 °C, Milli-Q, Millipore, Billerica, MA).
The analytical thin-layer chromatography (TLC) plates were
aluminum-backed ultrapure silica gel (Siliaplate, 60 Å pore size,
250 ␮m plate thickness, Silicycle, Quebec, QC). Flash column silica
gel was provided by Silicycle (Siliaflash Irregular Silica Gels F60,
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Price et al.
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Fig. 3. 1H NMR spectra of Bn-H3trenpa (7; 300 MHz, 25 °C, MeOD, top) and the In3+ complex In(Bn-trenpa) (13; 400 MHz, 25 °C, DMSO-d6,
bottom).
Table 1. Radiolabeling experiments with 111In and the novel acyclic ligands Bn-H3nonapa (3), H3nonapa (4), and
Bn-H3trenpa (7), and the previously published ligands H2dedpa, H4octapa, H5decapa, DOTA, and DTPA.22
Radiolabeling
conditions
HPLC tR
(min)b
Serum stability
1 h (%)c
Serum stability
24 h (%)c
Complex
RCY (%)a
[
[
[
[
[
[
[
[
111In(nonapa)]
111In(Bn-nonapa)]
111In(Bn-trenpa)]
111In(dedpa)]+
111In(octapa)]−
111In(decapa)]2−
111In(DOTA)]−
pH 4.5, 10 min, RT
pH 4.5, 45 min, RT
pH 4.5, 45 min, RT
pH 4.5, 10 min, RT
pH 4.5, 10 min, RT
pH 4.5, 10 min, RT
pH 4.5, 30 min, 90 °C
pH 4.5, 10 min, RT
97
93
15
99
99
95
97
99
5.0
8.9
10.5
5.9
4.7
5.4
3.5
6.5
93.5 0.4
27.0 0.9
NAd
96.1 0.1
93.8 3.6
89.7 1.6
89.6 2.1
86.5 2.2
44.5 25.9
16.6 0.4
NAd
19.7 1.5
92.3 0.1
89.1 1.7
89.4 2.2
88.3 2.2
111In(DTPA)]2−
aRCY = radiochemical yield.
btR = retention time.
cBlood serum stability results evaluated by PD-10 size-exclusion column elution.
dNA = not applicable.
using GE Healthcare Life Sciences PD-10 desalting columns (size
exclusion for molecular weight (MW) < 5000 Da; 1 Da = 1 g/mol)
and counted with a Capintec CRC 15R well counter. Radiolabeling
of DOTA with 111In was performed using a Biotage Initiator
microwave reactor (␮W). Analyses of radiolabeled complexes
were carried out using a Waters xbridge BEH130 C18 reverse phase
(150 mm × 6 mm) analytical column on a Waters Alliance HT 2795
separation module equipped with a Raytest Gabi Star NaI (Tl)
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8
Can. J. Chem. Vol. 92, 2014
Fig. 4. HPLC radiotrace of 111In(nonapa) (tR = 5.0 min, 97% RCY)
showing small impurities.
H]+: 284.2127; found [M + H]+: 284.2133, PPM (parts per million) =
2.1.
N,N== -[(Benzyl)-N,N=,N== -[(6-methoxycarbonyl)pyridine-2-
yl]methyl]diethylenetriamine (2)
To a solution of 1 (260 mg, 0.917 mmol) and sodium carbonate
(excess, ϳ500 mg) in dry acetonitrile (distilled over CaH2, 10 mL)
was added methyl-6-bromomethylpicolinate (synthesized accord-
ing to a literature procedure;22 654 mg, 2.84 mmol). The resulting
solution was stirred at 60 °C for 20 h under Ar. Sodium carbonate
was removed by filtration and the solvent was removed in vacuo.
The crude product was purified by silica chromatography (Com-
biFlash Rf automated column system; 40 g HP silica; A: dichlo-
romethane, B: methanol, A: gradient 100% to 20%) to afford 2 as a
yellow oil (541 mg, 0.741 mmol, 81%). 1H NMR (300 MHz, CDCl3) ␦:
7.90–7.88 (m, 3H), 7.67–7.65 (m, 4H), 7.20–7.13 (m, 10H, Bn), 3.91 (s,
9H), 3.75 (s, 4H, Pic-CH2-N), 3.71 (s, 2H, (Pic-CH2-N), 3.50 (s, 4H,
Bn-CH2-N), 2.54 (m, 8H, dien-H). 13C NMR (75 MHz, CDCl3) ␦: 165.47,
160.78, 146.72, 138.66, 136.99, 128.35, 127.94, 126.74, 126.50, 123.19,
60.77, 60.17, 58.89, 52.54, 51.81. HR-ESI-MS calcd. for [C42H46N6O6 +
H]+: 731.3557; found [M + H]+: 731.3549, PPM = –1.1.
Fig. 5. Mouse serum stability assay of 111In3+ complexes of ligands
from Table 1, determined after 1 and 24 h by PD-10 size-exclusion
column elution.
Bn-H3nonapa, N,N== -[(benzyl)-N,N=,N== -[(6-carboxy)pyridine-2-
yl]methyl]diethylenetriamine (3)
To a solution of 2 (375 mg, 0.513 mmol) in a mixture of tetrahy-
drofuran/deionized water (3:1, 5 mL) was added LiOH (300 mg). The
reaction mixture was stirred at ambient temperature for 2 h. A
portion of HCl was added (5 mL, 6 mol/L), and then the mixture
was reduced to dryness in vacuo. The mixture was dissolved in
deionized water (4 mL) and purified via semipreparative reverse-
phase (RP) HPLC (10 mL/min, gradient: A: 0.1% TFA (trifluoroacetic
acid) in deionized water, B: CH3CN; 5% to 100% B linear gradient in
25 min; tR = 16 min, broad). Product fractions were pooled, con-
centrated in vacuo, dissolved in HCl (5 mL, 6 mol/L), and then
concentrated in vacuo again to remove trifluoroacetic acid. The
HCl salt of 3, Bn-H3nonapa·4HCl·H2O, was obtained as a light
yellow solid (330.3 mg, 93%, using the molecular weight of the HCl
salt as determined by elemental analysis). 1H NMR (300 MHz,
MeOD) ␦: 8.05 (d, J = 7.8 Hz, 2H), 7.99–7.94 (m, 3H), 7.88 (t, J = 7.7 Hz,
1H), 7.54–7.42 (m, 3H), 7.38–7.30 (m, 10H), 4.66 (s, 4H), 4.48 (s, 4H),
3.84 (s, 2H), 3.54 (m, 4H, dien-H), 3.19 (m, 4H, dien-H). 13C NMR
(75 MHz, CDCl3) ␦: 165.85, 164.65, 151.69, 150.67, 146.87, 146.17,
145.73, 141.94, 139.81, 131.72, 130.33, 129.23, 129.13, 128.86, 128.42,
127.67, 125.84, 125.20, 59.72, 56.78, 56.35, 49.59, 49.48. HR-ESI-MS
calcd. for [C39H40N6O6 + H]+: 689.3088; found [M + H]+: 689.3077,
PPM = –1.5. Elemental analysis calcd. for Bn-H3nonapa·4HCl·H2O
(C39H40N6O6·4HCl·H2O = 852.562) (%): C 54.94, H 5.44, N 9.86;
found: C 55.21 (⌬ = 0.27), H 5.65 (⌬ = 0.21), N 9.54 (⌬ = 0.32).
detector and a Waters 996 photodiode array (PDA) detector. 111In
was cyclotron produced (Advanced Cyclotron Systems, Model
TR30) by proton bombardment through the reaction 111Cd(p,n)111In
and was provided by Nordion as 111InCl3 in 0.05 mol/L HCl.
N,N== -[Benzyl]diethylenetriamine (1)
H3nonapa, N,N=,N== -[(6-carboxy)pyridine-2-yl)methyl]-
Diethylenetriamine (5.0 mL, 46.3 mmol) was added to dry meth-
anol (distilled over CaH2, 100 mL), followed by benzaldehyde
(9.43 mL, 96.6 mmol), and the mixture was refluxed for 2 h under
Ar. The reaction mixture was cooled (0 °C) in an ice bath and
NaBH4 (12.2 g, 323 mmol) was added slowly and in small portions
to prevent boiling. The reaction mixture was stirred overnight for
ϳ16 h at ambient temperature. The reaction mixture was concen-
trated in vacuo and then a saturated aqueous solution of NaHCO3
(ϳ100 mL) and chloroform (200 mL) was added. The aqueous layer
was extracted twice more with dichloromethane (100 mL); the
combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo to afford a yellow oil. The crude product
was purified by silica chromatography (CombiFlash Rf automated
column system; 120 g HP silica; A: dichloromethane, B: methanol,
A: gradient 100% to 40%) to afford 1 as a yellow oil (8.15 g,
diethylenetriamine (4)
To a solution of 2 (60.0 mg, 0.0822 mmol) in glacial acetic acid
(10 mL) was added Pd/C (6 mg, ϳ10 wt%). Hydrogen gas was purged
and vented for 3 min through the reaction mixture, which was
then stirred under a hydrogen atmosphere (balloon) for 16 h. The
Pd/C was filtered out on a fine fritted glass filter and rinsed with
HCl (3 mol/L) and acetonitrile; the filtrate was then evaporated to
dryness in vacuo. The crude product was then deprotected with-
out further purification by adding HCl (10 mL, 12 mol/L) and re-
fluxing for 16 h. The reaction mixture was concentrated in vacuo,
dissolved in deionized water (4 mL) and purified via semiprepara-
tive reverse-phase HPLC (10 mL/min, gradient: A: 0.1% TFA (trifluo-
roacetic acid) in deionized water, B: CH3CN; 5% to 100% B linear
gradient in 25 min; tR = 8.1 min, broad). Product fractions were
pooled, concentrated in vacuo, dissolved in HCl (5 mL, 6 mol/L),
and then concentrated in vacuo again to remove trifluoroacetic
acid. The HCl salt of H3nonapa (4) was obtained as a light yellow
solid (17.3 mg, 47%). 1H NMR (400 MHz, D2O) ␦: 8.05–8.01 (m, 1H),
1
28.8 mmol, 62%). H NMR (300 MHz, CDCl3) ␦: 7.33–7.24 (m, 10H,
Bn-H), 3.80 (s, 4H, Bn-CH2-N), 2.75 (m, 8H, ethylene-H), 1.44 (s, 3H,
-NH-). 13C NMR (75 MHz, CDCl3) ␦: 140.17, 129.71, 129.06, 128.64,
128.28, 127.32, 55.12, 53.72, 44.68. HR-ESI-MS calcd. for [C18H25N3 +
Published by NRC Research Press

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Price et al.
9
7.91–7.86 (m, 3H), 7.84–7.80 (m, 2H), 7.72–7.70 (m, 1H), 7.47–7.45
(m, 2H), 4.36 (s, 4H), 4.10 (s, 2H), 3.44 (m, 4H), 3.21 (m, 4H). 13C NMR
(100 MHz, D2O) ␦: 168.00, 166.61, 163.27, 162.92, 157.85, 151.07,
147.73, 147.52, 146.89, 141.01, 139.61, 129.95, 129.50, 127.40, 126.69,
125.53, 125.12, 124.75, 57.09, 52.03, 50.59, 45.60. HR-ESI-MS calcd.
for [C25H28N6O6 + H]+: 509.2149; found [M + H]+: 509.2144, PPM =
–0.9.
NMR (75 MHz, MeOD) ␦: 167.09, 151.99, 151.86, 148.35, 148.17,
140.95, 140.83, 132.83, 132.74, 131.43, 131.40, 130.54, 130.41, 130.32,
128.75, 126.65, 126.42, 60.79, 60.36, 57.10, 56.90, 51.71. HR-ESI-MS
calcd. for [C48H51N7O6 + H]+: 822.3979; found [M + H]+: 822.3977,
PPM = –0.3. Elemental analysis calcd. for Bn-H3trenpa·6HCl
(C16H18N4O4·6HCl = 1040.097) (%): C 55.4, H 5.52, N 9.42; found: C
55.50 (⌬ = 0.10), H 5.83 (⌬ = 0.31), N 9.64 (⌬ = 0.22).
N,N=,N== -Tris[benzyl]ethylamine (5)
N,N== -[p-Nitrobenzyl]diethylenetriamine (8)
Tris(2-aminoethyl)amine (tren; 2.0 mL, 13.4 mmol) was added to
dry methanol (distilled over CaH2, 25 mL), followed by benzalde-
hyde (3.92 mL, 44.1 mmol), and the mixture was refluxed for 2 h
under Ar. The reaction mixture was cooled (0 °C) in an ice bath and
NaBH4 (4.10 g, 107 mmol) was added slowly and in small portions
to prevent boiling. The reaction mixture was stirred overnight for
ϳ16 h at ambient temperature. The reaction mixture was concen-
trated in vacuo and then a saturated aqueous solution of NaHCO3
(ϳ100 mL) and chloroform (100 mL) was added. The aqueous layer
was extracted twice more with dichloromethane (100 mL), the
combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo to afford a yellow solid. The crude product
was purified by silica chromatography (CombiFlash Rf automated
column system; 120 g HP silica; A: dichloromethane with 0.5%
triethylamine, B: methanol, A: gradient 100% to 50%) to afford 5 as
an orange oil (938 mg, 2.25 mmol, 17%). The product streaked very
badly on the column, and significant yield was lost. 1H NMR
(300 MHz, CDCl3) ␦: 7.25 (m, 15H, Bn-H), 3.71 (s, 6H, Bn-CH2-N), 3.24
(s, 3H, -NH), 2.64–2.56 (m, 12H, dien-H). 13C NMR (75 MHz, CDCl3) ␦:
139.14, 127.99, 127.82, 126.62, 53.55, 53.14, 46.44. HR-ESI-MS calcd.
for [C27H36N4 + H]+: 417.3018; found [M + H]+: 417.3018, PPM = 0.
Diethylenetriamine (0.775 mL, 7.18 mmol) was added to
dry methanol (distilled over CaH2, 15 mL), followed by p-4-
nitrobenzaldehyde (2.17 mg, 14.4 mmol), and then refluxed for 2 h.
The reaction mixture was cooled (0 °C) in an ice bath and NaBH4
(2.72 g, 71.8 mmol) was added slowly and in small portions to
prevent boiling. The reaction mixture was stirred overnight for
ϳ16 h at ambient temperature. The reaction mixture was concen-
trated in vacuo and then a saturated aqueous solution of NaHCO3
(ϳ50 mL) and chloroform (50 mL) was added. The aqueous layer
was extracted twice more with dichloromethane (50 mL), the com-
bined organic layers were dried over MgSO4, filtered, and concen-
trated in vacuo to afford an orange oil. The crude product was
purified by silica chromatography (CombiFlash Rf automated col-
umn system; 80 g HP silica; A: dichloromethane, B: methanol, A:
gradient 100% to 40%) to afford 8 as a yellow oil (1.07 g, 2.87 mmol,
1
40%). H NMR (300 MHz, CDCl3) ␦: 8.14–8.11 (m, 4H, NO2-Bn-H),
7.49–7.47 (m, 4H, NO2-Bn-H), 3.88 (s, 4H), 2.73 (s, 8H), 1.71 (s, 3H,
-NH-). 13C NMR (75 MHz, CDCl3) ␦: 148.30, 146.83, 128.46, 123.42,
53.03, 49.15, 48.86. HR-ESI-MS calcd. for [C18H23N5O4 + H]+:
374.1828; found [M + H]+: 374.1829, PPM = 0.2.
N,N== -[(p-Nitrobenzyl)-N,N=,N== -[(6-methoxycarbonyl)pyridine-
2-yl]methyl]diethylenetriamine (9)
N,N=,N== -Tris[benzyl]-tris[(6-methoxycarbonyl)pyridine-2-
yl)methyl]ethylamine (6)
To a solution of 8 (92.1 mg, 0.246 mmol) and sodium carbonate
(excess, ϳ300 mg) in dry acetonitrile (distilled over CaH2, 10 mL)
was added methyl-6-bromomethyl picolinate (synthesized accord-
ing to a literature procedure;22 170.2 mg, 0.739 mmol). The solu-
tion was stirred at 60 °C for 20 h under Ar. Sodium carbonate was
removed by filtration and the solvent was removed in vacuo. The
crude product was purified by silica chromatography (CombiFlash
Rf automated column system; 40 g HP silica; A: dichloromethane,
B: methanol, A: gradient 100% to 20%) to afford 9 as an orange oil
(130 mg, 0.158 mmol, 64%). 1H NMR (300 MHz, CDCl3) ␦: 8.08–8.04
(m, 4H), 7.95–7.91 (m, 3H), 7.76–7.71 (m, 2H), 7.65–7.57 (m, 3H),
7.44–7.40 (m, 5H), 3.95 (s, 6H, methyl ester), 3.94 (s, 3H, methyl
ester), 3.78 (s, 4H), 3.75 (s, 2H), 3.63 (s, 4H), 2.62–2.58 (m, 8H,
dien-H). 13C NMR (75 MHz, CDCl3) ␦: 165.50, 160.43, 159.89, 147.21,
146.96, 146.89, 137.37, 137.09, 129.02, 125.61, 123.65, 123.60, 123.50,
123.39, 123.31, 60.78, 60.34, 58.31, 52.80, 52.27. HR-ESI-MS calcd. for
[C42H44N8O10 + H]+: 821.3259; found [M + H]+: 821.3268, PPM = 1.1.
To a solution of 5 (299 mg, 0.717 mmol) and sodium carbonate
(excess, ϳ1 g) in dry acetonitrile (distilled over CaH2, 10 mL) was
added methyl-6-bromomethylpicolinate (synthesized according
to a literature procedure)22 (495 mg, 2.15 mmol). The solution was
stirred at 60 °C for 20 h under Ar. Sodium carbonate was removed
by filtration and the solvent was removed in vacuo. The crude
product was purified by silica chromatography (CombiFlash Rf
automated column system; 80 g HP silica; A: hexanes, B: ethyl
acetate, A: gradient 100% to 0%) to afford 6 as an orange oil
(394 mg, 0.456 mmol, 64%). 1H NMR (300 MHz, CDCl3) ␦: 7.96–7.93
(m, 3H), 7.72–7.70 (m, 6H), 7.28–7.18 (m, 15H), 3.97 (s, 9H, methyl
ester), 3.78 (s, 6H), 3.51 (s, 6H), 2.46 (m, 12H). 13C NMR (75 MHz,
CDCl3) ␦: 165.68, 161.09, 146.92, 138.99, 137.13, 128.51, 128.11,
126.89, 125.61, 123.34, 60.39, 59.04, 52.91, 52.73, 51.96. HR-ESI-MS
calcd. for [C51H57N7O6 + H]+: 864.4449; found [M + H]+: 864.4466,
PPM = 2.0.
Bn-H3trenpa, N,N=,N== -tris[benzyl]-tris[(6-carboxy)pyridine-2-
yl)methyl]ethylamine (7)
p-NO2-Bn-H3nonapa, N,N== -[(p-nitrobenzyl)-N,N=,N== -
[(6-carboxy)pyridine-2-yl]methyl]diethylenetriamine (10)
To a solution of 9 (130 mg, 0.158 mmol) in a mixture of tetrahy-
drofuran/deionized water (3:1, 5 mL) was added LiOH (150 mg). The
reaction mixture was stirred at ambient temperature for 2 h. A
portion of HCl was added (5 mL, 6 mol/L), and then the mixture
was reduced to dryness in vacuo. The mixture was dissolved in
deionized water (4 mL) and purified via semipreparative reverse-
phase HPLC (10 mL/min, gradient: A: 0.1% TFA (trifluoroacetic acid)
in deionized water, B: CH3CN; 5% to 100% B linear gradient in
25 min; tR = 18 min, broad). Product fractions were pooled, con-
centrated in vacuo, dissolved in HCl (5 mL, 6 mol/L), and then
concentrated in vacuo again to remove trifluoroacetic acid. The
product p-NO2-Bn-H3nonapa (10) was obtained as a light-orange
solid (20 mg, 16%). Due to instability and decomposition, elemen-
tal analysis and 13C NMR were not obtained. Further work with
To a solution of 6 (237 mg, 0.274 mmol) in a mixture of tetrahy-
drofuran/deionized water (3:1, 5 mL) was added LiOH (170 mg). The
reaction mixture was stirred at ambient temperature for 2 h. A
portion of HCl was added (5 mL, 6 mol/L), and then the mixture
was reduced to dryness in vacuo. The solid was dissolved in deion-
ized water (4 mL) and purified via semipreparative reverse-phase
HPLC (10 mL/min, gradient: A: 0.1% TFA (trifluoroacetic acid) in
deionized water, B: CH3CN; 5% to 100% B linear gradient in 25 min;
tR = 17.7 min, broad). Product fractions were pooled, concentrated
in vacuo, dissolved in HCl (5 mL, 6 mol/L), and then concentrated
in vacuo again to remove trifluoroacetic acid. The HCl salt of 7,
Bn-H3trenpa·6HCl, was obtained as a yellow solid (229 mg,
0.220 mmol, 80%, using the molecular weight of the HCl salt as
1
determined by elemental analysis). H NMR (300 MHz, MeOD) ␦:
1
8.08–7.96 (m, 6H), 7.53–7.43 (m, 9H), 7.36–7.31 (m, 9H), 4.55 (s, 6H),
this scaffold has not been attempted due to instability. H NMR
(300 MHz, D2O) ␦: 8.03–7.79 (m, 10H), 7.55–7.45 (m, 7H), 4.56 (s, 4H),
4.35 (s, 6H), 3.51 (m, 6H, ethylene-H), 3.13 (m, 6H, ethylene-H). 13
C
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Can. J. Chem. Vol. 92, 2014
4.41 (s, 4H), 3.81 (s, 2H), 3.48–3.44 (m, 4H, dien-H), 3.15–3.10 (m, 4H,
dien-H).
ϳ3–5 mCi for mouse serum competitions) was transferred into
each vial, allowed to react at ambient temperature for 10 min, and
then analyzed by RP-HPLC to confirm radiolabeling and calculate
yields. If the reaction was not complete, it was left for a 45 min
total reaction time and then checked again by RP-HPLC. Previ-
ously obtained radiolabeling yields and serum stability results for
the chelators DTPA, DOTA, H2dedpa, H4octapa, and H5decapa are
also presented and were performed under identical experimental
protocols at the same facilities (TRIUMF/Nordion).22 Areas under
the peaks observed in the radioactive HPLC trace were integrated
to determine radiolabeling yields. Elution conditions used for RP-
HPLC analysis were gradient: A: 10 mmol/L NaOAc buffer pH 4.5, B:
CH3CN; 0% to 100% B linear gradient 20 min. The radiometal com-
plex [111In(dedpa)]+ used a modified HPLC gradient of A: 10 mmol/L
NaOAc buffer pH 4.5, B: CH3CN; 0% to 5% B linear gradient 20 min.
[In(Bn-nonapa)] (11)
Bn-H3nonapa·4HCl·H2O (HCl salt of 3; 10.0 mg, 0.0117 mmol)
and In(ClO4)3·6H2O (8.00 mg, 0.0153 mmol) were dissolved in HCl
(aq; 1 mL, 0.1 mol/L) in a 20 mL screw cap vial. The pH was adjusted
to ϳ4.5 with NaOH (aq; 0.1 mol/L) with stirring. The reaction mix-
ture was stirred at 60 °C for 1 h, then evaporated to dryness to
afford [In(Bn-nonapa)] (11) as a white solid in a quantitative yield.
The reaction mixture turned cloudy white during the reaction, as
1
the insoluble and neutrally charged metal complex formed. H
NMR (600 MHz, DMSO-d6) ␦: 8.45–8.7.6 (broad m, 9H, pyr-H), 7.52–
7.23 (broad m, 10H, benzyl-H), 4.65–3.70 (broad m, 8H), 3.30–2.55
(broad m, 8H), 2.45–2.10 (broad m, 2H). 13C NMR (150 MHz, DMSO-
d6) ␦: 166.26, 164.19, 153.79, 153.26, 153.09, 148.05, 147.80, 147.73,
147.64, 147.54, 142.16, 139.26, 138.39, 138.06, 133.51, 132.19, 131.90,
131.70, 128.86, 128.68, 128.62, 128.42, 128.33, 128.25, 127.86, 127.10,
126.32, 126.03, 123.43, 123.04, 122.93, 122.70, 118.20, 64.11, 59.73,
57.98, 53.33, 52.26, 49.60, 49.47, 49.10, 48.89, 44.15. HR-ESI-MS
calcd. for [C39H37115InN6O6 + H]+: 801.1892; found [M + H]+:
801.1888, PPM = –0.5.
[
[
111In(Bn-nonapa)] tR = 8.9 min, [111In(nonapa)] tR = 5.0 min, and
111In(Bn-trenpa)] tR = 10.5 min.
Mouse serum stability challenge
The compounds [111In(Bn-nonapa)], [111In(nonapa)], and [111In(Bn-
trenpa)] were prepared with the radiolabeling protocol as de-
scribed above. Mouse serum was removed from the freezer and
allowed to thaw at ambient temperature for 30 min. In triplicate
for each 111In complex listed above, solutions were made in sterile
vials with 750 ␮L mouse serum, 500 ␮L of 111In complex (10 mmol/L
NaOAc buffer, pH 5.5), 250 ␮L phosphate buffered saline (PBS), and
were left to sit at ambient temperature. After 1 h, half of the
mouse serum competition mixture (750 ␮L) was removed from
each vial, diluted to a total volume of 2.5 mL with phosphate
buffered saline, and then counted in a Capintec CRC 15R well
counter to obtain a value for the total activity to be loaded on the
PD-10 column. The 2.5 mL of diluted mouse serum competition
mixture was then loaded onto a PD-10 column that had previously
been conditioned via elution with 20 mL of PBS. The 2.5 mL of
loading volume was allowed to elute into an 111In waste container,
and then the PD-10 column was eluted with 3.5 mL PBS and col-
lected into another sterile vial. This 3.5 mL of collected eluent,
which contained 111In bound/associated with serum proteins (size-
exclusion for MW < 5000 Da), was counted in a well counter, and
then compared to the total amount of activity that was loaded on
the PD-10 column to obtain the percentage of 111In that was bound
to serum proteins and therefore no longer chelate bound. The
percent stability values shown in Table 1 represent the percentage
of 111In that was retained on the PD-10 column and therefore still
chelate bound.
[In(nonapa)] (12)
H3nonapa (4; 14.9 mg, 0.0222 mmol) and In(ClO4)3·6H2O
(15.0 mg, 0.0288 mmol) were dissolved in HCl (aq; 1 mL, 0.1 mol/L)
in a 20 mL screw cap vial. The pH was adjusted to ϳ4.5 with NaOH
(aq; 0.1 mol/L) with stirring. The reaction mixture was stirred at
60 °C for 1 h, then evaporated to dryness to afford [In(nonapa)] (12)
as a white solid in a quantitative yield. No product was observed to
precipitate from the yellow reaction mixture. 1H NMR (600 MHz,
DMSO-d6) ␦: 8.21–7.75 (m, 8H, pyr-H), 7.38–7.31 (m, 1H, pyr-H), 4.77–
4.75 (m, 1H), 4.44–3.70 (m, 7H), 3.34–2.70 (m, 8H). 13C NMR
(150 MHz, DMSO-d6) ␦: 164.56, 163.57, 153.20, 152.44, 152.02, 146.56,
146.27, 141.69, 141.59, 141.15, 137.85, 129.06, 128.68, 127.54, 126.47,
125.26, 125.12, 123.90, 122.02, 121.94, 52.60, 51.84, 51.78, 51.00,
50.15, 49.99, 49.12, 44.27, 43.10, 29.89, 29.10. HR-ESI-MS calcd. for
[C25H25115InN6O6 + H]+: 621.0953; found [M + H]+: 621.0958, PPM =
0.8.
[In(Bn-trenpa)] (13)
Bn-H3trenpa·6HCl (HCl salt of 7; 41.5 mg, 0.0399 mmol) and
In(ClO4)3·6H2O (27.0 mg, 0.0519 mmol) were dissolved in HCl (aq;
1 mL, 0.1 mol/L) in a 20 mL screw cap vial. The pH was adjusted to
ϳ4.5 with NaOH (aq; 0.1 mol/L) with stirring. The reaction mixture
was stirred at 60 °C for 1 h, then evaporated to dryness to afford
[In(Bn-trenpa)] (13) as a white solid in a quantitative yield. The
reaction mixture turned cloudy white during the reaction, as the
Supplementary material
1H/13C NMR spectra of final synthesized compounds are avail-
able with the article through the journal Web site at http://
nrcresearchpress.com/doi/suppl/10.1139/cjc-2013-0542.
1
insoluble and neutrally charged metal complex formed. H NMR
(400 MHz, DMSO-d6) ␦: 8.33–7.85 (m, 8H), 7.44–7.18 (m, 16H), 4.93–
4.64 (broad m, 2H), 4.18–3.64 (broad m, 8H), 3.40–3.00 (broad m,
8H), 3.00–2.52 (broad m, 6H). 13C NMR (100 MHz, DMSO-d6) ␦:
166.16, 164.57, 163.82, 163.08, 158.99, 158.17, 153.86, 153.16, 147.81,
147.73, 147.56, 144.88, 144.46, 138.18, 137.64, 137.57, 131.67, 131.08,
129.10, 128.81, 128.75, 128.47, 128.29, 127.86, 127.33, 127.16, 127.10,
126.75, 126.20, 125.96, 125.85, 123.58, 123.43, 123.09, 122.65, 59.90,
58.65, 58.48, 58.33, 58.25, 57.98, 57.45, 57.38, 57.19, 50.45, 50.34,
48.01, 47.75. HR-ESI-MS calcd. for [C48H48115InN7O6 + H]+: 934.2783;
found [M + H]+: 934.2775, PPM = –0.9.
Acknowledgements
We acknowledge the Natural Sciences and Engineering
Research Council of Canada (NSERC) for Canada Graduate
Scholarships-Master's (CGS-M) and -Doctoral (CGS-D) fellowships
(E.W.P.) and many years of research support (C.O.), as well as the
University of British Columbia for a Four Year Doctoral Fellowship
(4YF) (E.W.P.). C.O. acknowledges the Canada Council for the Arts
for a Killam Research Fellowship (2011–2013) and the Alexander
von Humboldt Foundation for a Research Award, as well as Prof.
Dr. Peter Comba and his research group in Heidelberg for hospi-
tality and interesting discussions.
111In radiolabeling studies
The chelating ligands Bn-H3nonapa (3), H3nonapa (4), and Bn-
H3trenpa (7) were made up as stock solutions (1 mg/mL,
ϳ10−3 mol/L) in deionized water. An aliquot of each ligand stock
solution was transferred to screw cap mass spectrometry vials and
made up to 1 mL with pH 5.0 NaOAc (10 mmol/L) buffer to a final
concentration of ϳ365 ␮mol/L for each sample. An ϳ10 ␮L aliquot
of the 111InCl3 stock solution (ϳ1 mCi for labeling studies and
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