H4octapa: An acyclic chelator for 111In radiopharmaceuticals

Article abstract of DOI:10.1021/ja3024725

This preliminary investigation of the octadentate acyclic chelator H ₄octapa (N₄O₄) with ¹¹¹In/ ¹¹⁵In³⁺ has demonstrated it to be an improvement on the shortcomings of the current industry "gold standards" DOTA (N ₄O₄) and DTPA (N₃O₅). The ability of H₄octapa to radiolabel quantitatively ¹¹¹InCl₃ at ambient temperature in 10 min with specific activities as high as 2.3 mCi/nmol (97.5% radiochemical yield) is presented. In vitro mouse serum stability assays have demonstrated the ¹¹¹In complex of H ₄octapa to have improved stability when compared to DOTA and DTPA over 24 h. Mouse biodistribution studies have shown that the radiometal complex [¹¹¹In(octapa)]^- has exceptionally high in vivo stability over 24 h with improved clearance and stability compared to [ ¹¹¹In(DOTA)]^-, demonstrated by lower uptake in the kidneys, liver, and spleen at 24 h. ¹H/¹³C NMR studies of the [In(octapa)]^- complex revealed a 7-coordinate solution structure, which forms a single isomer and exhibits no observable fluxional behavior at ambient temperature, an improvement to the multiple isomers formed by [In(DTPA)]^2- and [In(DOTA)]^- under the same conditions. Potentiometric titrations have determined the thermodynamic formation constant of the [In(octapa)]^- complex to be log K_ML = 26.8(1). Through the same set of analyses, the [^111/115In(decapa)] ^2- complex was found to have nonoptimal stability, with H ₅decapa (N₅O₅) being more suitable for larger metal ions due to its higher potential denticity (e.g., lanthanides and actinides). Our initial investigations have revealed the acyclic chelator H ₄octapa to be a valuable alternative to the macrocycle DOTA for use with ¹¹¹In, and a significant improvement to the acyclic chelator DTPA.

Full text of DOI:10.1021/ja3024725

Article
pubs.acs.org/JACS
111
H4octapa: An Acyclic Chelator for In Radiopharmaceuticals
Eric W. Price,†,‡ Jacqueline F. Cawthray,†,‡ Gwendolyn A. Bailey,† Cara L. Ferreira,§ Eszter Boros,†,‡
Michael J. Adam,*,‡ and Chris Orvig*,†
†Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver,
British Columbia, Canada V6T 1Z1
‡TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 2A3
§Nordion, 4004 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 2A3
S
* Supporting Information
ABSTRACT: This preliminary investigation of the octaden-
tate acyclic chelator H4octapa (N4O4) with 111In/115In3+ has
demonstrated it to be an improvement on the shortcomings of
the current industry “gold standards” DOTA (N4O4) and
DTPA (N3O5). The ability of H4octapa to radiolabel
quantitatively 111InCl3 at ambient temperature in 10 min
with specific activities as high as 2.3 mCi/nmol (97.5%
radiochemical yield) is presented. In vitro mouse serum
stability assays have demonstrated the 111In complex of H4octapa to have improved stability when compared to DOTA and
DTPA over 24 h. Mouse biodistribution studies have shown that the radiometal complex [111In(octapa)]− has exceptionally high
in vivo stability over 24 h with improved clearance and stability compared to [111In(DOTA)]−, demonstrated by lower uptake in
1
the kidneys, liver, and spleen at 24 h. H/13C NMR studies of the [In(octapa)]− complex revealed a 7-coordinate solution
structure, which forms a single isomer and exhibits no observable fluxional behavior at ambient temperature, an improvement to
the multiple isomers formed by [In(DTPA)]2− and [In(DOTA)]− under the same conditions. Potentiometric titrations have
determined the thermodynamic formation constant of the [In(octapa)]− complex to be log KML = 26.8(1). Through the same set
of analyses, the [111/115In(decapa)]2− complex was found to have nonoptimal stability, with H5decapa (N5O5) being more
suitable for larger metal ions due to its higher potential denticity (e.g., lanthanides and actinides). Our initial investigations have
revealed the acyclic chelator H4octapa to be a valuable alternative to the macrocycle DOTA for use with 111In, and a significant
improvement to the acyclic chelator DTPA.
There are currently several 111In/90Y-based radiopharmaceu-
INTRODUCTION
■
111In is an important isotope in nuclear medicine for single
ticals FDA-approved for clinical use and many more in clinical
trials, demonstrating the importance of these isotopes in
photon emission computed tomography (SPECT) imaging and
nuclear medicine.7−13 The combination of 111In for imaging
performing dosimetry for therapeutic chelate-based radiophar-
and dosimetry and 90Y/177Lu for therapy is very effective but is
dependent on having a solid chelator foundation that can bind
both isotopes with exceptionally high stability (thermodynamic
and kinetic) and that has very similar biological behavior with
both isotopes (biologically equivalent).14−21 The macrocycle
DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid) is the industry “gold standard” for chelation of 111In,
90Y, and 177Lu (Chart 1); however, biovector conjugates suffer
from the need for elevated temperatures and extended reaction
times to achieve quantitative radiolabeling yields (typically 60−
95 °C, for 30−120 min), which is not optimal for use with heat-
sensitive biomolecules like antibodies.22−25 Peptide biovectors
can often be labeled at elevated temperatures without suffering
deleterious effects to their binding integrity and have also
shown excellent tumor targeting and uptake; however, with the
current chelator offerings they sometimes demonstrate high
maceuticals. 111In is a cyclotron-produced isotope (111Cd-
(p,n)111In) that decays with a half-life of ∼2.8 days via electron
capture (100% EC); it emits γ rays (245 and 172 keV) that can
be used for imaging, and Auger electrons that can be used for
therapy.1 One of the most promising radiotherapeutic isotopes,
90Y (t1/2 = ∼2.67 days), is essentially radiographically silent as it
only emits β− particles, and therefore must be used in
combination with an imaging isotope such as 111In, 89Zr, 86Y,
64Cu, or 67/68Ga to study its organ uptake and dosimetry.2−4
The generator-produced indium isotope 110mIn (t1/2 = 69 min)
is a very attractive positron emission tomography (PET)
imaging surrogate for 111In, and the two isotopes can be
seamlessly interchanged in bifunctional-chelate (BFC)-based
radiopharmaceuticals because they share identical chemical
properties.5 When used with the OctreoScan kit, 110mIn has
been shown to significantly improve spatial resolution,
dosimetry, and tumor identification when compared to 111In;5
however, the generator parent isotope 110Sn has a half-life of
∼4.1 h, which makes the generator life very short.6
Received: March 13, 2012
© XXXX American Chemical Society
A
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Chart 1. Structures of the 111In-Coordinating and Industry “Gold Standard” Chelators DTPA/CHX-A″-DTPA and DOTA, the
68Ga-Coordinating Chelator H2dedpa, and the Novel Entrants H4octapa and H5decapa
and persistent kidney uptake that can disrupt the predictive
power of dosimetry calculations and cause unwanted radiation
exposure.20,26
acetic acid (referred to herein as H4octapa) (Chart 1),36 and
the novel decadentate derivative H5decapa. DOTA and DTPA
were used as benchmarks of the industry “gold standards” for
111In chelation.
Since the properties of the radiometal−chelate complex can
strongly influence the biodistribution profile of its peptide
conjugate, a new chelator with an improved clearance profile
and decreased persistent kidney uptake would be a welcome
improvement. Although the influence of chelate properties on
the biodistribution of BFC conjugates is less significant for large
biovectors like antibodies (∼150 kDa for an intact antibody)
than it is for smaller peptides, the difference can still be
significant.2,20,21 Antibody biovectors show exceptionally high
tumor uptake and have long biological half-lives (2−3 weeks27),
and thus are well matched with the long half-lives of 90Y (∼2.67
days) and 177Lu (∼6.6 days).1 The acyclic chelator DTPA
(diethylenetriamine tetraacetic acid) will successfully radiolabel
these isotopes in 10−15 min at ambient temperature; however,
the in vivo stability is less than optimal, showing a greater
extent of decomposition and nontarget organ uptake than
DOTA.20,28−30 The DTPA derivative CHX-A″-DTPA has an
improved stability profile with 86/90Y when compared to DTPA,
while retaining its low-temperature labeling abilities; however,
the stability and in vivo kinetic inertness are still not as good as
DOTA, and its In3+ complexes have been less studied.28−32
Despite the poor in vivo stability, DTPA is the chelator used in
the currently available FDA approved 111In-based radiophar-
maceuticals.
Our group has recently investigated a series of acyclic
chelators, resulting in identification of the promising 67/68Ga
chelator H2dedpa (Chart 1).33,34 Initial radiolabeling experi-
ments, in vitro apo-transferrin stability experiments, and
biodistribution studies in mice have demonstrated H2dedpa
to be an ideal chelator for 67/68Ga-based radiopharmaceuticals.35
This success has prompted the investigation of larger acyclic
frameworks based on the basic H2dedpa scaffold, supporting
higher denticities for accommodating larger radiometal ions
such as 111In, 90Y, 177Lu, 89Zr, and 225Ac. Herein we report the
synthesis, characterization, coordination chemistry, thermody-
namic stability, radiolabeling, in vitro mouse serum stability,
and in vivo biodistribution studies of the 111In complexes of the
previously reported but uniquely applied octadentate chelator
N,N′-bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N′-di-
RESULTS AND DISCUSSION
■
Although the macrocycle DOTA is the most stable chelator for
111In, it is essentially incompatible with antibodies due to its
required high-temperature radiolabeling, and the excellent
match of antibodies with long-lived therapeutic isotopes
makes this mismatch especially unfortunate. This suggests an
important and high-priority need for new, highly stable acyclic
chelators for use with isotopes such as 111In, 90Y, and 177Lu,
especially when conjugated to antibodies. There is a need for
chelators that combine the exceptional in vivo stability and
kinetic inertness of DOTA with the fast ambient temperature
labeling kinetics of DTPA. These observations suggest that the
target for an ideal 111In/90Y/177Lu chelator would have the
following properties. It should (1) be acyclic or if macrocyclic,
be constructed with free-moving appendages to emulate the
low-temperature acyclic radiolabeling kinetics, such as that of
BCNOTA.37 The coordination complex should (2) be very
stable, both thermodynamically and kinetically, but with top
priority placed on exceptional in vivo kinetic inertness,
particularly to demetalation and/or transchelation. The
coordination complex should (3) exhibit minimal isomerization
and fluxional behavior, and the chelator should be isomerically
pure with preferably only one stable isomer being formed and
administered or, like CHX-A″-DTPA, the superior isomer of
the chelator isolated and administered pure.28−30 Finally, it
should (4) ideally have similar properties in its chelate/
bifunctional-chelate complexes with both of the radiometal ions
to be used in an imaging/therapy pair (i.e., 111In/90Y), so that
biological clearances and organ uptakes are sufficiently similar
and that accurate dosimetry information can be obtained
(bioequivalence). Not every one of these four points must be
met for a chelator to be useful as a BFC for radiometal ions,
and especially point number four may be hard to achieve,
considering the different coordination numbers and geometries
preferred by different metal ions such as 111In (6−8 coordinate)
and 90Y (8−9 coordinate).38
B
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a
Scheme 1. Synthesis of Compounds 1, 2, 3, 4, 5, 6, and H4octapa
a
Reagents and conditions: a) NaBH4 (2.4 equiv), CH3OH, 4 h, 0 °C, 51%; b) PBr3 (1.1 equiv), CHCl3, 0 °C, 3.5 h, 99%; c) CH3OH, NaBH4 (4.3
equiv), 8 h, 49%; d) CH3CN, Na2CO3 (excess), 60 °C, Ar (g), 12−16 h, 4 = alkylation with tert-butyl bromoacetate, 93%, 6 = alkylation with 2, 39%;
e) AcOH, Pd/C (10 wt %), H2 (g), 12 h, 87%; f) HCl (6 M), Δ 8 h, 75%.
a
Scheme 2. Synthesis of Compounds 7, 8, 9, 10, and H5decapa
a
Reagents and conditions: a) CH3OH, NaBH4 (7 equiv), 8 h, 40%; b) CH3CN, Na2CO3 (excess), 60 °C, Ar (g), 12−16 h, 8 = alkylation with tert-
butyl bromoacetate, 66%, 10 = alkylation with 2, 45%; c) AcOH, Pd/C (5 wt %), H2 (g), 12 h, 30%; d) HCl (6 M), Δ 8 h, 71%.
Synthesis and Characterization. We have studied a
number of novel acyclic chelators, and herein we report our two
most promising candidates for use with 111In. The previously
reported, but here uniquely applied, octadentate chelator N,N′-
bis(6-carboxy-2-pyridylmethyl)ethylenediamine-N,N′-diacetic
acid (H4octapa) was previously used with various paramagnetic
lanthanides to assess their abilities as MRI contrast agents.36 As
an expansion of this scaffold, the novel decadentate derivative
H5decapa has also been synthesized and evaluated. The
chelators H4octapa and H5decapa were synthesized with a
general reaction scheme that follows N-benzyl protection, N-
alkylation with an alkyl halide, benzyl deprotection via
hydrogenation, a second alkyl halide N-alkylation, and finally
deprotection in refluxing HCl (6 M) (Schemes 1 and 2).
Previous methods to synthesize the similar acyclic chelator
H2dedpa utilized reductive amination reactions;33,36 however,
this method resulted not only in reduction of the imines but
also reduction of the picolinic acid methyl ester groups to
carboxylates and alcohols, which were very difficult to separate
in subsequent steps, resulting in low purified yields. The
synthetic scheme presented here circumvents this problem by
avoiding the use of sodium borohydride in the presence of the
picolinic acid moiety (Schemes 1 and 2). The only significant
problem encountered with these syntheses is the lability of the
picolinate groups to hydrogenation. Benzylated intermediates
that contain picolinate groups undergo unwanted cleavage
during hydrogenation, and to minimize this undesirable
pathway the reaction order was optimized to perform the
debenzylation step only in the presence of the tert-butylacetate
groups (Schemes 1 and 2). The obvious drawback to this
method is that after hydrogenation, the tert-butylacetate-
alkylated ethylenediamine/diethylenetriamine compounds (5/
9) are not UV active and must be stained with iodine to be
visualized. Hydrogenation of the diethylenetriamine scaffold
during the synthesis of H5decapa resulted in substantial
cleavage of the diethylenetriamine backbone, resulting in
lower yields than the equivalent hydrogenation of the
ethylenediamine scaffold in the H4octapa synthesis. Decreasing
C
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1
Figure 1. H NMR spectra in D2O at ambient temperature of (top) H4octapa (300 MHz) and [In(octapa)]− (600 MHz) showing simple
diastereotopic splitting due to minimal isomerization and (bottom) H5decapa (400 MHz) and [In(decapa)]2− (600 MHz) showing complicated
splitting arising from multiple isomers.
the Pd/C catalyst loading appeared to slow down reaction
kinetics and did not improve selectivity for debenzylation over
the unwanted side reactions.
(yields for both compounds using previous methods were
under 1%). The incorporation of multiple synthons in the
synthesis of these chelators will allow for more straightforward
syntheses of bifunctional derivatives via (4-nitrobenzyl)-
ethylenediamine/diethylenetriamine backbone derivatives,
which should be an improvement over the difficult and tedious
synthesis of bifunctional macrocycles like p-isothiocyanatoben-
zyl DOTA.39
The presented synthetic schemes are an obvious improve-
ment over previous attempts, allowing for improved yields and
the purification of all intermediates despite the large number of
polar functional groups; however, the problem of nonselective
debenzylation and ethylene backbone cleavage of diethylenetri-
amine is problematic, and with larger scaffolds such as tris(2-
aminoethyl)amine (TREN) this problem becomes more severe.
These challenges suggest that new protecting-group chemistry
must be employed to further improve the synthesis of these
picolinic acid-based chelators and facilitate the elaboration of
larger scaffolds and novel bifunctional derivatives. The relatively
simple five-step synthesis of H4octapa is achieved with a
cumulative yield of ∼12%, and the five-step synthesis of
H5decapa was completed with a cumulative yield of ∼2.5%
In general, chelates/BFC that exhibit minimal isomerization
are preferred and tend to be more stable in vivo.40−42 The H
1
NMR spectrum of [In(octapa)]− showed clear and sharply
resolved diastereotopic splitting of the protons associated with
both picolinic acid moieties (methylene-H, 4.49 ppm, 2J = 16.1
2
Hz, and 4.34 ppm, J = 16.1 Hz), as well as one of the acetic
2
acid arms (3.27 ppm, J = 19.3 Hz), suggesting a 7-coordinate
solution structure (Figure 1). The coupling constants observed
2
for these metal-bound appendages were J = 16.1 Hz for the
D
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Figure 2. DFT structure of [In(octapa)]− (solvent = water) showing an 8-coordinate structure (left), and the electrostatic potentials of the complex
between octapa4− with In3+ mapped onto the electron density (right). The MEP represents a maximum potential of 0.03 au, and a minimum of
−0.25 au, mapped onto electron density isosurfaces of 0.002 e Å−3 (red to blue = negative to positive).
2
two picolinic acid methylene groups, and J = 19.3 Hz for the
acetic acid arm. The second acetic acid arm appeared as a broad
doublet (3.19 ppm) with a much smaller coupling constant of
2J = 8.4 Hz, showing diasterotopic splitting but suggesting it is
not metal-bound and does not form a five-membered
metallocycle like the other metal-bound appendages. The
sharp and clear diasterotopic splitting seen for [In(octapa)]−
suggests the presence of a single isomer with no observable
fluxional interconversion at ambient temperature (on the NMR
time scale). No change in the number of peaks in the 13C NMR
spectrum of the ligand H4octapa was observed upon In3+
coordination, suggesting no chemically distinct isomers were
formed (different coordination number/polarity) (Figure S8,
Supporting Information [SI]). The HPLC radiotrace of
[In(octapa)]− showed a single sharp peak, affirming that the
radiometal complex exhibits minimal isomerization (Figure S19
[SI]). This reveals a very stable and inert coordination structure
present as a single isomer, unlike the [In(DOTA)]− complex
which shows multiple isomers with rapid fluxional isomer-
ization at ambient temperature.42,43
free ligand.44 These observations for [111In(DTPA)]2− suggest
that little or no fluxional behavior between isomers/
diastereomers occurs in solution at ambient temperature (on
the NMR time scale), although multiple isomers are indeed
formed.42,44,45 If minimal isomerization and fluxional behavior
are optimal for stability,40−42 then the NMR solution structures
of the currently investigated In3+ chelator complexes would
have them ranked as [In(octapa)]− > [111In(DTPA)]2−
>
[In(DOTA)]− > [In(decapa)]2−. As previously discussed,
DTPA has been shown to be inferior to DOTA in terms of
in vivo stability with In3+ and so does not fit with this trend;
however, this work largely follows the trend with the in vivo
stability of H4octapa being superior to DOTA, and H5decapa
being inferior to both.
The H NMR spectrum of the hexadentate [In(dedpa)]+
1
complex in D2O was very similar to that of [In(octapa)]+, and
showed a sharp doublet for each set of methylene protons (4.58
ppm, 2J = 17.3 Hz, and 4.13 ppm, 2J = 17.4 Hz) associated with
1
the picolinic acid moiety (Figure S26 [SI]). The H NMR
spectrum of the hexadentate [In(dedpa)]+ complex in DMSO-
d6 revealed two sharp doublets for each set of methylene
protons (4.27 ppm, 2J = 16.6 Hz, 4.26 ppm, 2J = 16.3 Hz, 3.90
The In3+ complex formed with the potentially decadentate
chelator H5decapa, [In(decapa)]2−, displayed a more complex
1H NMR splitting (Figure 1) and a large number of additional
13C NMR peaks that were not observed for the free ligand
H5decapa (Figures S12, S14, SI). This observation, along with
the presence of two peaks in the HPLC radiotrace (tR = 5.4 min
(5%), 7.7 min (95%), Figure S20 [SI]) suggests that multiple
isomers (including chemically distinct isomers) of [In-
(decapa)]2− are present in solution at ambient temperature.
Considering that In3+ typically forms 7−8-coordinate com-
plexes, the decadentate chelator H5decapa may have several
unbound carboxylates, which could give rise to different
protonation species in solution and could explain the two
peaks observed in the HPLC radiotrace. Additionally, NMR
experiments (D2O) and HPLC experiments were performed at
different pH, which could influence the protonation species
present in solution. [In(DTPA)]2− displays a complicated
splitting of the 1H NMR peaks in a similar fashion to
[In(decapa)]2−, and also yields sharp splitting patterns
suggesting that little or no fluxional behavior between multiple
isomers/diastereomers occurs in solution at ambient temper-
ature.44,45 Unlike [In(decapa)]2−, [In(DTPA)]2− retains a
simple 13C NMR spectrum with no additional peaks to the
2
2
ppm, J = 16.6 Hz. 3.89 ppm, J = 16.6 Hz) attached to the
picolinic acid moieties, suggesting clean diastereotopic splitting
arising from two separate isomers, most likely from DMSO
binding to the open coordination sites of In3+ (Figure S26 [SI).
The sharp peaks observed for [In(dedpa)]+ in D2O and
DMSO-d6 suggest no observable fluxional behavior at ambient
temperature, and the presence of only one set of peaks in the
13C NMR spectrum (same as unbound H2dedpa, Figures S16
and S18 [SI]) and one peak in the HPLC radiotrace (vide
infra) suggests the formation of only one isomer. In
consideration of these results, the very simple and sharp
diastereotopic proton splitting observed in the 1H NMR
spectrum of [In(octapa)]−, coupled with the single sharp peak
observed in the HPLC radiotrace, suggests that this complex
displays the least amount of isomerization and fluxional
behavior in solution at ambient temperature out of the
currently investigated novel chelators and the industry “gold
standards” DTPA and DOTA.
DFT Structures and Molecular Electrostatic Potential
Maps. Although solid-state structures of metal−chelate
complexes obtained from X-ray crystallography are useful,
E
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Figure 3. DFT structure of [In(decapa)]2− (solvent = water) showing an 8-coordinate structure, with one picolinic acid group unbound and
extended away from the metal center (left), and the electrostatic potentials of the complex between decapa5− with In3+ mapped onto the electron
density (right). The MEP represents a maximum potential of 0.03 au, and a minimum of −0.25 au, mapped onto electron density isosurfaces of
0.002 e Å−3 (red to blue = negative to positive).
they are often not representative of the solution-phase
structures, and so DFT calculations (modeled in water) and
NMR studies in D2O of solution structures are most relevant to
a discussion of potential in vivo applications.38,42,46 The DFT
structure of [In(octapa)]− (Figure 2, left) reveals an 8-
coordinate complex with approximately C2v symmetry, showing
tight binding of In3+ with slight puckering of the picolinic acid
moieties. The solution structure of [In(octapa)]− as deduced
by NMR spectroscopy suggests a 7-coordinate structure with
one acetic acid arm unbound; however, it is not certain from
the broad doublet (3.22 ppm) and smaller coupling constant
(8.4 Hz) of the unbound acetic acid arm whether or not there is
transient metal binding. The DFT structure of [In(decapa)]2−
(Figure 3, left) shows an 8-coordinate structure, with one
picolinic acid and all three acetic acid arms bound to indium (as
well as the three tertiary backbone nitrogen atoms), and one
picolinic acid group unbound and extended away from the
metal center. This unsymmetric coordination sphere has a large
impact on the electrostatic potential of the complex (Figure 3,
right), with the MEP map revealing the entire molecular surface
to be more electronegative than [In(octapa)]− (Figure 2, right).
[In(decapa)]2− shows areas of very high electronegative
potential around the unbound picolinic acid group, most likely
from the unbound deprotonated carboxylate group (Figure 3,
right). The unsymmetric high density of electronegative
potential shown on the MEP map of [In(decapa)]2−, in
contrast to the symmetric and less electronegative charge
distribution of [In(octapa)]−, may result in a higher propensity
toward protonation and protein binding in vivo, and may be
partially responsible for the poor stability in vivo.
Radiolabeling Experiments. Initial radiolabeling experi-
ments demonstrated the ability of H4octapa to radiolabel
quantitatively 111In at ambient temperature in 10 min, showing
a single sharp peak in the HPLC radiotrace at tR = 4.7 min
(Figure S27, SI). Radiolabeling with [In(octapa)]− yields
specific activities as high as 2.3 mCi/nmol (∼5 mCi/μg, 2300
mCi/μmol) in 10 min at ambient temperature. DOTA was
radiolabeled with the same activity of 111In (∼1 mCi) at the
same ligand concentration (10−7 M) used to obtain the specific
activity listed above for [In(octapa)]−, after 10 min at ambient
temperature less than 40% 111In was radiometalated. This
demonstrates the ability of H4octapa to radiolabel 111In
quantitatively and rapidly in high specific activities at ambient
temperature, which is in sharp contrast to the “gold standard”
DOTA. The theoretical maximum 111In specific activity has
been calculated to be ∼46 mCi/nmol, and specific activities as
high as 22 mCi/nmol have been reported for 111In-labeled
DOTA−peptide conjugates under ideal conditions; however,
temperatures of 100 °C for 30 min were required, and specific
activities that high are not typical with DOTA-conjugates.47 To
compare recently reported examples, a 111In-CHX-A″-DTPA−
Re(Arg11)CCMSH peptide conjugate yielded a specific activity
of ∼0.29 mCi/nmol,48 DTPA−(gp100:154−162mod) (HLA-
A2.1 binding peptide conjugate) showed a maximum specific
activity of ∼0.35 mCi/nmol,49 and a DOTA−RGD conjugate
obtained a specific activity as high as ∼0.050 mCi/nmol;50
these are more typical values and are much lower than the ∼2.3
mCi/nmol specific activity obtained for [In(octapa)]− during
this study. It must be considered, however, that the examples
listed above are for chelator−peptide conjugates, and the values
obtained in this study were for nonconjugated [In(octapa)]−.
DOTA−biovector conjugates typically offer lower specific
activities when compared to acyclic chelators such as DTPA
and CHX-A″-DTPA.48 The HPLC radiotrace of [In(decapa)]2−
was less promising and revealed two peaks (tR = 5.4 min (5%),
7.7 min (95%)), possibly due to multiple chemically distinct
isomers being formed, with specific activities being low at
∼0.030 mCi/nmol. As previously discussed, DOTA required
temperatures of 80 °C in a microwave reactor for ∼20 min to
label 111In.
Thermodynamic Stability. Formation constants (log
ML) are well-established measurements of thermodynamic
K
stability for metal−ligand complexes and are typically reported
in the literature; however, pM (−log[Mn+free]) values are much
more accurate figures for predicting in vivo thermodynamic
stability under physiologically relevant conditions. Values of pM
are calculated (at specific conditions, here [Mn+] = 1 μM, [Lx−]
= 10 μM) to take into account all of ligand basicity (ligand pKa
values), free metal concentration, ligand-to-metal ratios, pH,
and metal hydroxide formation. The thermodynamic stability of
[In(octapa)]− was determined to be log KML = 26.8(1) (pM =
26.5), which is significantly higher than that for DOTA (log
KML = 23.9(1), pM = 18.8) and similar to DTPA (log KML
=
29.0, pM = 25.7) but shows the highest pM value of all three
In3+ complexes (Table 1). Although CHX-A″-DTPA has been
evaluated with Y3+ to be very stable,51 it has not been
thoroughly studied with In3+ and to our knowledge formation
constants have not yet been reported. Despite [In(DOTA)]−
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a
Table 1. Formation Constants (log KML) and pM Values for
Table 2. Data from Mouse Serum Stability Challenges
In3+ Complexes
Performed at Ambient Temperature (n = 3), with Stability
Shown As the Percentage of Intact 111In Complex
a
chelator
log KML
pM
dedpa2−
26.60(4)
26.8(1)
27.56(5)
35.4
25.9
26.5
23.1
34.1
25.7
18.8
18.7
complex
1 h stability (%)
24 h stability (%)
octapa4−
[
[
[
[
[
111In(dedpa)]+
111In(octapa)]−
111In(decapa)]2−
111In(DOTA)]−
111In(DTPA)]2−
96.1 0.1
93.8 3.6
89.7 1.6
89.6 2.1
86.5 2.2
19.7 1.5
92.3 0.04
89.1 1.7
89.4 2.2
88.3 2.2
decapa5−
oxine (tris)53
DTPA52,53
DOTA53,54
transferrin55
29.0
23.9(1)
18.3
a
Calculated for 10 μM total ligand and 1 μM total metal at pH 7.4 and
25 °C.
being significantly more stable in vivo than is [In-
(DTPA)],2−20,28−30 the log KML and pM values are much
lower for DOTA than for DTPA with In3+. It is interesting to
note that the trend of stability constants (log KML) and pM
values in Table 1 do not correlate well with in vivo stability,
which emphasizes that these thermodynamic parameters are
not the only factors involved in determining biological stability.
The kinetic parameters of ligand−metal on/off rates are the
most important factor, with [In(DOTA)]− having one of the
lowest pM values and very high stability/kinetic inertness in
vivo, and with In(oxine)3 having the highest log KML and pM
values (log KML = 35.3, pM = 34.1) and dissociating very
quickly in vivo.13 These values reinforce the concept that in
vitro competition experiments such as mouse serum and apo-
transferrin competitions, and in vivo biodistribution and
stability studies are essential to evaluate the practical in vivo
kinetic inertness and metabolism of radiometal complexes.
Because the thermodynamic formation constants of the
H4octapa, H5decapa, and H2dedpa ligands with In3+ are all
very similar, the large differences in pM values must be a result
of ligand basicity. Despite these complications, it is encouraging
that [In(octapa)]− has an exceptionally high pM value of 26.5,
which is higher than the values previously for determined
DOTA (18.8) and DTPA (25.7) with In3+.52−54
Stability Studies. In consideration of the fact that in vivo
kinetic inertness plays a crucial role in determining stability,
competition experiments using native biological chelators such
as those contained in blood serum (e.g., apo-transferrin,
albumin) are useful in vitro assays for predicting the in vivo
stability and kinetic inertness of radiometal ion complexes.
During preliminary experiments, mouse serum stability assays
were found to show transchelation of In3+ more aggressively
than were apo-transferrin assays. These mouse serum
competition experiments (incubated at ambient temperature)
have demonstrated [In(octapa)]− to have marginally higher
stability than [In(DOTA)]− and [In(DTPA)]2− after 24 h,
within error (92.3
0.04%, 89.4
2.2%, 88.3
2.2%,
respectively) (Table 2). [In(decapa)]2− also demonstrated
exceptional stability against mouse serum transchelation with a
stability of 89.1 1.7% at 24 h.
Figure 4. Biodistribution % ID/g values for [111In(DOTA)]−,
111In(octapa)]−, and [111In(decapa)]2−, with error bars plotted as
Mouse biodistribution studies were performed with [111In-
(octapa)]−, [111In(DOTA)]−, and [111In(decapa)]2−, and the
data summarized in Figure 4 (y-axis adjusted to 2% injected
dose (ID)/g for visualization of 4 and 24 h time points) shows
rapid clearance through the kidneys for [111In(octapa)]− and
[
standard deviations (note the y-axis set to 2.0% ID/g, for clarity of the
low activity 4 and 24 h time points).
[
111In(DOTA)]−, with activity clearing quickly from all organs.
that is unstable in vivo and undergoes demetalation/trans-
chelation, which is surprising because of the high degree of
stability of [111In(decapa)]2− in mouse serum competition
experiments (Table 2). The most promising result from these
The clearance of [111In(decapa)]2− is not as rapid, with 111In
levels slowly increasing over 24 h in the liver, spleen, and bone
(Table 3). These results are typical of a metal−ligand complex
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Table 3. Decay-Corrected % ID/g Values from the Biodistribution of 111In-Complexes in Healthy Female ICR Mice (6-8 weeks
old), n = 4, bold = Passed Student’s T-Test (p < 0.05)
organ
15 min
SD
1 h
SD
4 h
SD
24 h
SD
[
111In(DOTA)]−
spleen
kidney
intestine
liver
0.564
6.275
0.638
0.518
0.967
2.218
0.090
0.527
0.826
2.768
306.197
0.140
3.652
0.173
0.172
0.228
0.776
0.013
0.152
0.119
0.536
120.471
0.074
1.175
0.088
0.093
0.077
0.134
0.015
0.040
0.095
0.177
523.638
0.030
0.054
0.935
0.055
0.064
0.034
0.053
0.010
0.028
0.077
0.038
3.726
0.013
0.259
0.019
0.025
0.005
0.007
0.001
0.005
0.023
0.008
3.643
0.043
0.664
0.047
0.061
0.017
0.018
0.001
0.010
0.044
0.008
0.131
0.007
0.108
0.008
0.002
0.004
0.009
0.0005
0.004
0.011
0.002
0.069
0.631
0.047
0.033
heart
0.042
lung
0.095
brain
0.010
muscle
femur
blood
urine
0.025
0.063
0.116
542.402
111In(octapa)]−
[
spleen
kidney
intestine
liver
0.462
5.738
0.619
0.811
0.775
2.003
0.073
0.435
0.785
2.422
506.246
0.113
1.027
0.085
0.235
0.259
0.175
0.008
0.248
0.154
0.523
146.573
0.072
1.327
0.109
0.182
0.066
0.111
0.019
0.028
0.079
0.132
229.086
0.024
0.072
0.654
0.064
0.053
0.045
0.047
0.014
0.034
0.064
0.035
5.961
0.004
0.078
0.020
0.009
0.006
0.003
0.001
0.003
0.009
0.005
5.761
0.020
0.189
0.041
0.025
0.014
0.014
0.003
0.007
0.047
0.010
0.124
0.008
0.019
0.007
0.003
0.002
0.007
0.002
0.002
0.012
0.002
0.045
0.907
0.080
0.051
heart
0.033
lung
0.088
brain
0.004
muscle
femur
blood
urine
0.019
0.035
0.068
200.871
111In(decapa)]2−
[
spleen
kidney
intestine
liver
0.567
10.380
0.843
0.049
2.095
0.107
0.040
0.170
0.082
0.007
0.126
0.124
0.980
91.598
0.455
10.210
0.513
0.457
0.671
1.146
0.055
0.378
1.410
2.207
53.949
0.170
0.525
7.554
0.541
0.838
0.691
1.347
0.065
0.396
1.772
1.999
60.685
0.187
0.928
0.136
0.149
0.188
0.373
0.014
0.070
0.193
0.463
66.949
0.661
3.681
0.647
0.934
0.376
0.566
0.036
0.256
1.950
0.518
0.666
0.085
0.539
0.113
0.175
0.093
0.117
0.004
0.045
0.121
0.115
0.513
2.835
0.172
0.790
0.255
heart
1.123
0.272
lung
2.375
0.691
brain
0.071
0.033
muscle
femur
blood
urine
0.692
0.203
1.453
0.790
2.456
0.795
318.209
13.363
animal experiments is that the radiometal complex [111In-
(octapa)]− has exceptional in vivo stability over 24 h, with
improved clearance compared to [111In(DOTA)]− (Figure 4),
most notably from the kidneys (0.189 0.019% ID/g vs 0.664
0.108% ID/g, respectively, Figure S23 [SI]), liver (0.0248
0.0030% ID/g vs 0.0605 0.0022% ID/g, respectively, Figure
S24 [SI]), and spleen (0.0202 0.0083% ID/g vs 0.0426
0.0067% ID/g, respectively, Figure S25 [SI]) at 24 h (p < 0.05,
Table S4 [SI]). These findings are significant because in vivo
instability resulting in demetalation or transchelation via serum
proteins typically results in high uptake of “free” 111In in the
liver, spleen, bone, and kidneys (typical of transferrin-bound 3+
metals), with levels increasing over time.56 Surprisingly, despite
the exceptional mouse serum stability of [In(decapa)]2−, the in
vivo stability in mice was found to be suboptimal, with slower
clearance from the blood pool and most notably high persistent
kidney (3.68 0.54% ID/g) and increasing bone uptake (1.95
0.12% ID/g) over 24 h; however, all other organs contained
less than 1% ID/g after 24 h (Figure 4, Table 3). Although
these values are inadequate when compared to the in vivo
stability and clearance of exceptionally stable chelators like
compared to the very unstable 111In-citrate complex, which
has shown values of 18.7 3.7% ID/g in the kidneys after 24
h.57
Often 111InCl3 and 111In-citrate are used to emulate the
conditions of an unstable chelator that would undergo
complete and rapid decomposition/transchelation in vivo.
One commonly cited study (Ando et al.) has shown 111InCl3
to have higher liver, spleen, and kidney uptake than 111In-
citrate;56 however, another study has shown the kidney uptake
of 111InCl3 at 24 h in healthy rats to be 2.58 0.83% ID/g,58
which is significantly less than the value for 111In-citrate cited
above of 18.7 3.7% ID/g,57 and contradicts the observations
from Ando et al.56 Additionally, the highly anionic 111In-citrate
clears much more quickly through the kidneys than 111InCl3,
demonstrating that the hypothesis of rapid and complete
dissociation upon introduction of these radiometal species into
an animal is not accurate.56 Considering these inconsistencies,
it is important to utilize internal standards (such as [111In-
(DOTA)]− in this study), and additionally there is a need for
more clear and reliable baseline data for the biodistribution of
111In in its most commonly used forms.56,59,60
[
111In(DOTA)]− and [In(octapa)]−, it is still fair when
H
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In biological systems, transchelated indium is nearly all
bound to transferrin, with a maximum stability constant of log
KML = 18.3.55 Demetalation and hydroxide formation is a
significant concern for acidic metal ions like Ga3+ and In3+, and
a conditional stability constant for transferrin that takes
hydrolysis into account has been calculated as being ∼10.0
for In3+, which is higher even than Ga3+ at 6.9 and close to Fe3+
at 11.4.55 This demonstrates the strong competition that
transferrin holds for In3+ in vivo; however, the ligand exchange
kinetics of In3+ are quite slow and it forms a more inert
complex with transferrin than do Ga3+ and Fe3+.55,61 It is most
likely that, due to the high stability and inertness of complexes
like [In(DOTA)]− and the slow exchange kinetics with
transferrin, only gradual in vivo decomplexation is observed
over long periods such as >24 h. The higher levels of 111In in
the kidneys, liver, and spleen observed for [In(DOTA)]− after
24 h is most likely a result of this gradual exchange process, and
the improved inertness and clearance observed for [111In-
(octapa)]− correlates well with its lower isomerization and
higher pM value of 26.8(1) ([In(DOTA)]− pM = 18.8) (Table
1) and suggests superior kinetic inertness. Additionally, the
lower levels of 111In in the kidneys at 24 h seen for
[In(octapa)]−, being roughly a third of that of [In(DOTA)]−,
are very promising because persistent kidney uptake is observed
with many peptide conjugates of DOTA, CHX-A″-DTPA, and
especially DTPA.2,20,21,28−30,48 High residual activity in the
kidneys can decrease image quality and obstruct delineation of
tumors, decrease the accuracy of dosimetry, and deliver harmful
doses of radiation to the healthy and nontargeted kidneys when
used with therapeutic isotopes such as 90Y.
[In(DOTA)]− and [In(DTPA)]2− (18.8 and 25.7, respec-
tively). The same set of experiments and analyses was
performed with the potentially decadentate chelator H5decapa
(N5O5) and its [In(decapa)]2− complex; however, the
formation of multiple isomers observed via HPLC radiotraces
and NMR studies was not optimal, and unfavorable organ
uptake and clearance were observed in biodistribution studies
performed in mice. These results for H5decapa suggest that it is
not a suitable candidate for 111In radiopharmaceutical
elaboration; however, it may be a suitable candidate for
coordinating lanthanide and actinide radiometal ions that can
accommodate higher denticities, such as 90Y, 177Lu, and 225Ac.
The relatively simple five-step synthesis of H4octapa is achieved
with a cumulative yield of ∼12%, and the five-step synthesis of
H5decapa was completed with a cumulative yield of ∼2.5%.
Optimization of reaction conditions and/or exploration of
alternative protecting groups may improve these yields. Our
initial investigations have revealed the acyclic chelator H4octapa
to be a valuable alternative to the macrocycle DOTA and the
acyclic chelator DTPA by showing improved stability and
kinetic inertness, as well as fast ambient temperature radio-
labeling kinetics. Pending the results of investigations with 90Y
and 177Lu, H4octapa could present a very strong alternative to
DOTA as an ideal chelator for incorporation into radiometal-
based radiopharmaceuticals.
EXPERIMENTAL SECTION
■
Materials and Methods. All solvents and reagents were
purchased from commercial suppliers (TCI America, Sigma Aldrich,
Fisher Scientific) and were used as received. Mouse serum was
purchased frozen from Sigma Aldrich. DOTA was purchased from
Macrocyclics. The acyclic chelator H2dedpa was synthesized according
to the literature.33,36 The analytical thin-layer chromatography (TLC)
plates used were aluminum-backed ultrapure silica gel 60 Å, 250 μm
thickness; the flash column silica gel (standard grade, 60 Å, 32−63
mm) was provided by Silicycle. 1H and 13C NMR spectra were
recorded at ambient temperature on Bruker AV300, AV400, or AV600
instruments; the NMR spectra are expressed on the δ scale and were
referenced to residual solvent peaks and/or internal tetramethylsilane.
13C NMR experiments run in D2O were externally referenced to a
sample of CH3OD/D2O. Low-resolution mass spectrometry was
performed using a Waters ZG spectrometer with an ESCI electro-
spray/chemical-ionization source, and high-resolution electrospray-
ionization mass spectrometry (ESI-MS) was performed on a
Micromass LCT time-of-flight instrument at the Department of
Chemistry, University of British Columbia. Microanalysis for C, H, and
N was performed on a Carlo Erba Elemental Analyzer EA 1108. IR
spectra were collected neat in the solid state on a Thermo Nicolet
6700 FT-IR spectrometer. 111In(chelate) mouse serum stability
experiments were analyzed using GE Healthcare Life Sciences PD-
10 desalting columns (size exclusion for MW < 5000 Da) and counted
with a Capintec CRC 15R well counter. Radiolabeling of DOTA with
111In was performed using a Biotage Initiator microwave reactor (μW).
The HPLC system used for analysis and purification of cold
compounds consisted of a Waters 600 controller, Waters 2487 dual
wavelength absorbance detector, and a Waters delta 600 pump.
Phenomenex synergi hydro-RP 80 Å columns (250 mm × 4.6 mm
analytical and 250 mm × 21.2 mm semipreparative) were used for
purification of several of the deprotected chelators. Analysis of
radiolabeled complexes was carried out using a Waters xbridge
BEH130 C18 reverse phase (150 mm × 6 mm) analytical column
using a Waters Alliance HT 2795 separation module equipped with a
Raytest Gabi Star NaI (Tl) detector and a Waters 996 photodiode
array (PDA) detector. 111InCl3 was cyclotron produced and provided
by Nordion as a ∼0.05 M HCl solution.
CONCLUSIONS
■
Preliminary investigations of the octadentate acyclic chelator
H4octapa (N4O4) with 111In/In3+ have demonstrated it to be a
significant improvement on the shortcomings of the current
industry gold standards, DOTA (N4O4) and DTPA (N3O5).
Four major points were identified in the discussion as
guidelines to be used in selecting an ideal chelating agent for
radiometal ions, and [In(octapa)]− has been shown to be
superior to [In(DOTA)]− and [In(DTPA)]2− for a majority of
these points. We have demonstrated the ability of H4octapa to
quantitatively radiolabel 111In at ambient temperature, which, in
contrast to DOTA, allows it to be effectively used with sensitive
biovectors like antibodies. H4octapa has been radiolabeled with
111In in 10 min at ambient temperature with specific activities as
high as 2.3 mCi/nmol (97.5% radiochemical yield). In vitro
mouse serum stability assays have demonstrated H4octapa to
have slightly improved stability with 111In compared to that of
DOTA and DTPA over 24 h, within error. Mouse
biodistribution studies have shown that the radiometal complex
[
111In(octapa)]− has exceptionally high in vivo stability and
kinetic inertness, and compared to those of [111In(DOTA)]−,
[
111In(octapa)]− has improved stability and improved clearance
from the kidneys, liver, and spleen at 24 h. H/13C NMR
studies of the [In(octapa)]− complex have revealed a 7-
coordinate solution structure, which forms a single isomer and
exhibits no observable fluxional behavior at ambient temper-
ature on the NMR time scale and is an improvement on the
isomerization observed with [In(DTPA)]2− and the fluxional
isomerization of [In(DOTA)]−.40−42 Potentiometric titrations
have determined the thermodynamic formation constant of the
[In(octapa)]− complex to be log KML = 26.8(1) (pM = 26.5),
which reveals a higher pM value than those determined for
1
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Methyl 6-(Hydroxymethyl)picolinate (1). 1 was synthesized
according to a modified literature protocol.62 To a suspension of
dimethylpyridine-2,6-dicarboxylate (10.0 g, 51.2 mmol) in methanol
(400 mL), cooled in a salted ice bath, was slowly added sodium
borohydride (5.56 g, 146.9 mmol, 2.4 equiv) over a period of 1 h,
where the reaction mixture turned pink upon the addition of sodium
borohydride. The reaction mixture was stirred and kept on ice for 4 h.
After 4 h, TLC analysis revealed that the reaction mixture contained
starting material (Rf: 0.45, TLC in 100% EtOAc), product (Rf: 0.33),
and doubly reduced product (Rf: 0.24). The reaction mixture was
quenched regardless of the remaining starting material in order to
prevent over-reduction. The solution was diluted using dichloro-
methane (200 mL) and then quenched using saturated NaHCO3
(∼200 mL). The aqueous and organic layers were separated, and
much of the methanol from the aqueous phase was evaporated in
vacuo. The aqueous layer was then extracted with chloroform (2 × 100
mL) and ethyl acetate (3 × 100 mL). The combined organic layers
were dried (MgSO4), filtered, and concentrated in vacuo to dryness.
The resulting white solid was purified by column chromatography
(column 10” L × 2” W, eluted with a gradient of 3:1 ethyl acetate/
petroleum ether to 100% ethyl acetate) to afford the product as a
N,N′-(Benzyl)-N,N′-[(tert-butoxycarbonyl)methyl]-
ethylenediamine (4). To a solution of 3 (1.0 g, 4.2 mmol) and
sodium carbonate (1.76 g, 16.6 mmol, 3.9 equiv) in dry acetonitrile
(distilled over CaH2, 80 mL) was added dropwise a solution of tert-
butyl bromoacetate (1.31 mL, 8.88 mmol, 2.1 equiv). The reaction
mixture was stirred at 60 °C for 16 h. TLC (5% CH3OH in
dichloromethane) showed quantitative conversion of 3 to the tertiary
diamine. Sodium carbonate was filtered and the filtrate concentrated in
vacuo to dryness. The resulting yellow oil was purified by column
chromatography (column 2” L × 2” W, eluted with a gradient of 100%
petroleum ether to 10% ethyl acetate in petroleum ether) to afford 4 as
a yellow solid (1.85 g, 93%, Rf: 0.59 in 5% CH3OH in dichloro-
1
methane). H NMR (400 MHz, CDCl3): δ 7.40−7.29 (m, 10H, Bn-
H), 3.85 (s, 4H, Bn-CH2−N), 3.30 (s, 4H, (CH3)3CO−C(O)−CH2−
N), 2.88 (s, 4H, ethylene-H), 1.52 (s, 18H, (CH3)3CO−C(O)−CH2−
N). 13C NMR (100 MHz, CDCl3): δ 170.80, 139.13, 128.83, 128.08,
126.85, 80.51, 58.30, 55.06, 51.60, 28.11. HR-ESI-MS calcd for
[C28H40N2O4 + H]+: 469.3066; found [M + H]+: 469.3055.
N,N′-[(tert-Butoxycarbonyl)methyl]ethylenediamine (5). To
a solution of 4 (776.3 mg, 1.656 mmol) in glacial acetic acid (7 mL)
was added Pd/C (78 mg, ∼10 wt %). Hydrogen gas was bubbled
through the solution for 3 min, and then the reaction mixture was
stirred under a hydrogen atmosphere (balloon) for 16 h. The Pd/C
was filtered out over Celite, rinsing well with methanol, and the filtrate
was evaporated to dryness in vacuo. The crude product was purified by
silica gel column chromatography (eluted with a gradient of 100%
dichloromethane to 5% methanol in dichloromethane) and identified
by TLC using an I2 chamber to stain. Product fractions were combined
and concentrated in vacuo to afford the product 5 as a waxy yellow
1
white solid (4.35 g, 51%, Rf: 0.33 in 100% EtOAc). H NMR (300
MHz, CDCl3): δ = 7.95 (d, 1H, pyr-H), 7.79 (t, 1H, pyr-H), 7.55 (d,
1H, pyr-H), 4.83 (s, 2H, methylene-H), 4.31 (s, 1H, -OH), 3.92 (s,
3H, methyl-H). 13C NMR (75 MHz, CDCl3): δ 165.44, 160.62,
146.69, 137.57, 123.95, 123.54, 64.57, 52.72. HR-ESI-MS calcd for
[C8H9NO3 + H]+: 168.0661; found [M + H]+ 168.0658.
Methyl 6-(Bromomethyl)picolinate (2). 2 was synthesized
according to a modified literature protocol.62 To a solution of 1
(4.00 g, 23.9 mmol) in chloroform (100 mL) under argon at 0 °C was
added phosphorus tribromide (2.50 mL, 26.3 mmol, 1.1 equiv) in
chloroform (10 mL) dropwise over 15 min via dropping funnel. A
white precipitate formed, and then the solution turned bright yellow.
The dropping funnel was rinsed using chloroform (15 mL), and the
reaction mixture was stirred at 0 °C and monitored by TLC. A
miniworkup using sodium carbonate in water and ethyl acetate was
required to see reaction progress by TLC. At 3.5 h TLC indicated the
quantitative conversion of alcohol to alkyl halide. The reaction mixture
was quenched using sodium carbonate in water (75 mL) and was
extracted with chloroform (4 × 25 mL). The combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo to dryness
to afford an off-white solid. The crude product was purified through a
short silica plug (2” L × 2” W, 25% EtOAc in petroleum ether) to
afford 2 as an off-white solid (5.55 g, >99%, Rf: 0.64 in 30% petroleum
ether in EtOAc). 1H NMR (400 MHz, CDCl3): δ 8.01 (d, 1H, pyr-H),
7.84 (t, 1H, pyr-H), 7.65 (d, 1H, pyr-H), 4.61 (s, 2H, methylene-H),
3.96 (s, 3H, methyl-H). 13C NMR (100 MHz, CDCl3): δ 165.07,
157.22, 147.36, 138.16, 127.03, 124.31, 53.93, 32.91. HR-ESI-MS calcd
for [C8H8NO279Br + H]+: 229.9817; found [M + H]+: 229.9812.
N,N′-(Benzyl)ethylenediamine (3). To a solution of ethylenedi-
amine (2.78 mL, 41.6 mmol) in dry methanol (distilled over CaH2,
100 mL) was added benzaldehyde (8.48 mL, 83.2 mmol, 2 equiv). The
solution was refluxed for 4 h and then cooled via an ice bath. Addition
of NaBH4 (6.77 g, 179 mmol, 4.3 equiv) was performed slowly and in
small portions to prevent boiling, and the reaction mixture was stirred
for 4 h until completion. The solvent was evaporated in vacuo, and
then saturated NaHCO3 (∼50 mL), water (∼50 mL) and chloroform
(200 mL) were added. The aqueous layer was extracted twice more
with chloroform (100 mL). The combined organic layers were dried
over MgSO4, filtered, and concentrated in vacuo to afford a waxy,
yellow solid. Product was purified by silica column chromatography
(column 10” L × 2” W, eluted with a gradient of 100%
dichloromethane to 25% CH3OH in dichloromethane) to afford 3
as yellow oil (4.90 g, 49%, Rf: 0.33 in 20% CH3OH in dichloro-
1
solid (0.51 g, 87%, Rf: 0.20 in 10% CH3OH in dichloromethane). H
NMR (300 MHz, CDCl3): δ 3.37 (s, 4H, (CH3)3CO−C(O)−CH2−
N), 2.68 (s, 4H, ethylene-H), 2.03 (s, 2H, −NH−), and 1.43 (s, 18H,
(CH3)3CO−C(O)−CH2−N). 13C NMR (75 MHz, CDCl3): δ 171.67,
80.96, 51.46, 48.76, 28.01. HR-ESI-MS calcd for [C14H28N2O4 + H]+:
289.2127; found [M + H]+: 289.2126.
N , N ′ - [ ( t e r t - B u t o x y c a r b o n y l ) m e t h y l ] - N , N ′ - { [ 6 -
(methoxycarbonyl)pyridin-2-yl]methyl}ethylenediamine (6).
To a solution of 5 (431.5 mg, 1.496 mmol) and 2 (725 mg, 3.142
mmol, 2.1 equiv) in dry acetonitrile (distilled over CaH2, 20 mL) was
added sodium carbonate (∼300 mg). The solution was heated for 16 h
at 60 °C under argon. Sodium carbonate was removed by filtration and
rinsed with acetonitrile. The filtrate was concentrated in vacuo to
dryness, and the resulting yellow oil was purified by column
chromatography (eluted with a gradient of 100% dichloromethane
to 5% methanol in dichloromethane) to afford 6 as colorless oil (342
1
mg, 39%, Rf: 0.40 in 10% CH3OH in dichloromethane). H NMR
(300 MHz, CDCl3): δ 7.85−7.82 (m, 2H, pyr-H), 7.65−7.63 (m, 4H,
pyr-H), 3.85 (s, 4H, Pyr-CH2−), 3.83 (s, 6H, −O−CH3), 3.16 (s, 4H,
(CH3)3CO−CO−CH2−N), 2.67 (s, 4H, ethylene-H), 1.28 (s, 18H,
(CH3)3CO−C(O)−CH2−N). 13C NMR (75 MHz, CDCl3): δ 170.16,
165.39, 160.34, 146.74, 137.13, 125.84, 123.19, 80.66, 60.17, 55.98,
52.44, 52.09, 27.73. HR-ESI-MS calcd for [C30H42N4O8 + H]+:
587.3081; found [M + H]+: 587.3091.
H4octapa·4HCl·2H2O. N,N′-Bis(6-carboxy-2-pyridylmethyl)-
ethylenediamine-N,N′-diacetic Acid. A portion of 6 (277.4 mg,
0.4728 mmol) was dissolved in HCl (6 M) and refluxed for 8 h. White
solid was observed to precipitate from hot HCl (aq) below ∼60 °C.
The solution was then cooled in the freezer for 1 h. The product was
filtered and washed with cold ethanol and diethyl ether to afford the
HCl salt H4octapa as a white solid (265 mg, 75% yield using the
1
molecular weight of the HCl salt from elemental analysis). H NMR
(300 MHz, D2O): δ 8.12−8.02 (m, 4H, pyr-H), 7.72−7.69 (d, 2H,
pyr-H), 4.53 (s, 4H, Pyr-CH2−N), 3.97 (s, 4H, HOOC−CH2−N),
3.57 (s, 4H, ethylene-H). 13C NMR (75 MHz, D2O): δ 170.65, 165.57,
151.46, 145.43, 142.90, 128.83, 126.36, 58.07, 55.22, 51.48. IR (neat,
ATR-IR): ν = 1720.9 cm−1 (CO), 1635.9/1618.2 cm−1 (CC py).
HR-ESI-MS calcd for [C20H20N4O8 + H]+: 445.1359; found [M +
H]+: 445.1368. Elemental analysis: calcd % for H4octapa·4HCl·2H2O
(C20H20N4O8·4HCl·2H2O = 628.283): C 38.23, H 4.81, N 8.92;
found: C 38.34, H 4.81, N 8.93.
1
methane). H NMR (400 MHz, CDCl3): δ 7.42 (d, 8H, Bn-H), 7.34
(m, 2H, Bn-H), 3.86 (s, 4H, Bn-CH2−N), 2.83 (s, 4H, ethylene-H),
1.72 (s, 2H, amine-H). 13C NMR (100 MHz, CDCl3): δ 140.17,
127.94, 127.70, 126.45, 53.50, 48.39. HR-ESI-MS calcd for [C16H20N2
+ H]+: 241.1705; found [M + H]+: 241.1703.
J
dx.doi.org/10.1021/ja3024725 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Na[In(octapa)]. Portions of H4octapa·4HCl·2H2O (9.9 mg, 0.019
mmol) and In(ClO4)3·8H2O (11.7 mg, 0.021 mmol, 1.1 equiv) were
dissolved in HCl (aq) (1 mL, 0.1 M) in a 1 dram screw-cap vial. The
pH was adjusted to ∼4.5 with NaOH (aq) (0.1 M) while stirring. The
reaction mixture was stirred at 60 °C for 4 h, then evaporated to
N′). 13C NMR (75 MHz, CDCl3): δ 170.45, 170.78, 80.76, 80.67,
55.56, 54.03, 51.51, 47.17, 28.05, 27.98. HR-ESI-MS calcd for
[C22H43N3O6 + H]+: 446.3230; found [M + H]+: 446.3239.
N,N″-{[6-(Methoxycarbonyl)pyridin-2-yl]methyl}-N,N′,N″-
[(tert-butoxycarbonyl)methyl]diethylenetriamine (10). To a
solution of 9 (101.8 mg, 0.228 mmol) and 2 (110.4 mg, 0.479
mmol, 2.1 equiv) in dry acetonitrile (distilled over CaH2, 10 mL) was
added sodium carbonate (∼100 mg). The solution was heated for 16 h
at 60 °C under argon. Sodium carbonate was removed by filtration and
rinsed with acetonitrile. The filtrate was concentrated in vacuo to
dryness, and the resulting yellow oil was purified by column
chromatography (100% dichloromethane to 5% CH3OH in dichloro-
methane) to afford 10 as colorless oil (75.7 mg, 45%, Rf: 0.27 in 2.5%
1
dryness to afford Na[In(octapa)] as a white solid. H NMR (600
MHz, D2O): δ 8.34−8.32 (m, 2H, pyr-H), 8.23 (d, 2H, pyr-H), 7.85
(d, 2H, Pyr-H), 4.49 (d, 2H, Pyr-CH2−N, 2J = 16.1 Hz), 4.34 (d, 2H,
2
2
Pyr-CH2−N, J = 16.1 Hz), 3.27 (d, 2H, HOOC−CH2−N, J = 19.3
2
Hz), 3.19−3.18 (broad d, 2H, HOOC−CH2−N, J = 8.4 Hz), 3.07−
3.04 (m, 4H, ethylene-H). 13C NMR (150 MHz, D2O): δ 177.25,
167.84, 153.68, 148.21, 144.21, 128.27, 124.57, 59.39, 58.35, 55.24.
HR-ESI-MS calcd for [C20H18115InN4O8 + 2·Na]+: 602.9959; found
[M + 2·Na]+: 602.9942.
1
CH3OH and 2.5% triethylamine in dichloromethane). H NMR (300
MHz, CDCl3): δ 7.96−7.94 (m, 2H, Pyr-H), 7.82−7.70 (m, 4H, Pyr-
H), 3.97 (s, 4H, Pyr-CH2−N), 3.96 (s, 6H, −O−CH3), 3.28 (s, 4H,
(CH3)3CO−CO−CH2−N/N″), 3.23 (s, 2H, (CH3)3CO−CO−CH2−
N′), 2.72 (s, 8H, ethylene-H), 1.41 (s, 18H, (CH3)3CO−CO−CH2−
N/N″), 1.37 (s, 9H, (CH3)3CO−CO−CH2−N′). 13C NMR (75 MHz,
CDCl3): δ 170.66, 170.45, 170.37, 165.80, 160.93, 147.06, 137.29,
126.01, 123.45, 80.91, 80.85, 80.64, 60.52, 56.36, 56.29, 55.77, 52.74,
56.68, 55.77, 52.74, 52.68, 52.62, 52.42, 28.07. HR-ESI-MS calcd for
[C38H57N5O10 + H]+: 744.4184; found [M + H]+: 744.4199.
H5decapa·5HCl·2.5H2O. N,N″-{[6-(Carboxy)pyridin-2-yl]-
methyl}diethylenetriamine-N,N′,N″-triacetic Acid. A portion of
10 (76.7 mg, 0.1031 mmol) was dissolved in HCl (6 M) and refluxed
for 8 h. The reaction mixture was concentrated in vacuo to an off-
white powder, which was then purified by reverse-phase HPLC
(gradient: A: 0.1% TFA (trifluoroacetic acid), B: CH3CN; 0 to 100% B
linear gradient 25 min. tR = broad, 13.2−15 min). The HPLC fractions
were combined, 2 mL of HCl (6 M) was added, and the solvent was
removed in vacuo to drive off residual trifluoroacetic acid. Another 2
mL of HCl (6 M) was added and then concentrated in vacuo to afford
the HCl salt of H5decapa as a white solid (56 mg, 71% using molecular
weight from elemental analysis). 1H NMR (400 MHz, D2O): δ 8.11−
8.04 (m, 4H, Pyr-H), 7.72−7.71 (m, 2H, Pyr-H), 4.55 (s, 4H, Pyr-
CH2−N), 3.98 (s, 4H, HOOC−CH2−N/N″), 3.71 (s, 2H, HOOC−
CH2−N′), 3.44 (s, 4H, ethylene-H), 3.28 (s, 4H, ethylene-H). 13C
NMR (100 MHz, D2O): δ 172.54, 170.22, 168.18, 151.08, 145.92,
142.33, 128.81, 126.33, 57.89, 54.96, 54.27, 51.99, 50.82. IR (neat,
ATR-IR): ν = 1727.2 cm−1 (CO), 1642.7 cm−1 (CC py). HR-
ESI-MS calcd for [C24H29N5O10 + H]+: 548.1993; found [M + H]+:
548.1987. Elemental analysis: calcd % for H5decapa·5HCl·2.5H2O
(C24H29N5O10·5HCl·2.5H2O = 774.856): C 37.20, H 5.07, N 9.04;
found: C 37.28, H 4.91, N 8.72.
N,N″-(Benzyl)diethylenetriamine (7). To a solution of dieth-
ylenetriamine (5 mL, 46.2 mmol) in dry methanol (distilled over
CaH2, 100 mL) was added benzaldehyde (9.43 mL, 96.56 mmol, 2
equiv). The solution was refluxed for 4 h, and then cooled (0 °C) via
ice bath. Addition of NaBH4 (12.26 g, 323 mmol, 7 equiv) was
performed slowly and in small portions to prevent boiling. The
reaction solution was stirred for 4 h until completion. The solvent was
evaporated in vacuo, and then saturated NaHCO3 (∼100 mL) and
chloroform (200 mL) were 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 gel
column chromatography (column 16” L × 3” W, eluted with a gradient
of 2 to 10% CH3OH and 2% triethylamine in dichloromethane) to
afford 7 as yellow oil (5.23 g, 40%, Rf: 0.30 in 20% CH3OH in
1
dichloromethane). H NMR (300 MHz, CDCl3): δ 7.34−7.26 (m,
10H, Bn-H), 3.80 (s, 4H, Bn-CH2−N), 2.75 (s, 8H, ethylene-H), 1.94
(s, 3H, −NH−). 13C NMR (75 MHz, CDCl3): δ 140.54, 128.64,
128.34, 127.11, 54.08, 49.39, 48.92. HR-ESI-MS calcd for [C18H25N3 +
H]+: 284.2127; found [M + H]+: 284.2124.
N,N″-(Benzyl)-N,N′,N″-[(tert-butoxycarbonyl)methyl]-
diethylenetriamine (8). To a solution of 7 (673.3 mg, 2.37 mmol)
and sodium carbonate (excess, 450 mg) in dry acetonitrile (distilled
over CaH2, 20 mL) was added dropwise tert-butyl bromoacetate
(1.071 mL, 7.25 mmol, 3.05 equiv) under argon. The solution was
stirred at 60 °C for 20 h. Sodium carbonate was filtered and the filtrate
concentrated in vacuo to dryness. The resulting yellow oil was purified
by column chromatography (column 10” L × 2” W, eluted with a
gradient of 1 CH3OH to 10% CH3OH in dichloromethane) to afford
8 as light-yellow oil (974.4 mg, 66%, Rf: 0.36 in 10% CH3OH in
1
dichloromethane). H NMR (400 MHz, CDCl3): δ 7.41−7.29 (m,
Na2[In(decapa)]. A portion of H5decapa (8.6 mg, 0.0112 mmol)
and In(ClO4)3·8H2O (6.9 mg, 0.0124 mmol, 1.1 equiv) was dissolved
in HCl (aq) (1 mL, 0.1 M) in a 1 dram screw-cap vial. The pH was
adjusted to ∼4.5 with NaOH (aq) (0.1 M) while stirring. The reaction
mixture was stirred at 60 °C for 16 h and then evaporated to dryness
10H, Bn-H), 3.86 (s, 4H, Bn-CH2−N), 3.39 (s, 2H, (CH3)3CO−CO−
CH2−N′), 3.31 (s, 4H, (CH3)3CO−CO−CH2−N/N″), 2.84 (s, 8H,
ethylene-H), 1.54 (s, 18H, (CH3)3CO−CO−CH2−N/N″), 1.50 (s,
9H, (CH3)3CO−CO−CH2−N′). 13C NMR (100 MHz, CDCl3): δ
170.86, 170.69, 139.06, 128.79, 128.03, 126,82, 80.45, 80.39, 58.24,
55.88, 55.05, 52.45, 51.89, 28.08, 28.03. HR-ESI-MS calcd for
[C36H55N3O6 + H]+: 626.4169; found [M + H]+: 626.4166.
1
to afford Na2[In(decapa)]. H NMR (600 MHz, D2O): δ 8.33−7.71
(m, 6H, Pyr-H), 4.87−2.57 (m, 18H, complex diastereotopic
splitting). 13C NMR (150 MHz, D2O): δ 178.51, 178.35, 178.04,
177.42, 176.63, 168.43, 167.83, 154.01, 153.63, 148.54, 146.64, 145.44,
145.02, 144.17, 143.22, 142.83, 142.56, 128.77, 128.26, 128.12, 128.01,
124.42, 123.61, 123.53, 123.07, 61.29, 59.50, 59.31, 59.06, 58.32,
57.82, 55.71, 55.29, 53.99, 52.55, 52.46, 51.02. HR-ESI-MS calcd for
[C24H24115InN5O10 + H + 2Na]+: 704.0436; found [M + H + 2Na]+:
704.0430.
N,N′,N″-[(tert-Butoxycarbonyl)methyl]diethylenetriamine
(9). To a solution of 8 (702.6 mg, 1.12 mmol) in glacial acetic acid (10
mL) was added Pd/C (35 mg, ∼5 wt %). Hydrogen gas was bubbled
through the solution for 3 min, and then the reaction mixture was
stirred under a hydrogen atmosphere (balloon) for 16 h. The Pd/C
was filtered over Celite, rinsing with methanol, and then the filtrate
was evaporated to dryness in vacuo. The crude product was purified by
silica gel column chromatography (0 to 10% methanol in dichloro-
methane) and identified by TLC using an I2/silica chamber to stain.
Product fractions were combined and concentrated in vacuo to
dryness to afford the product 9 as colorless oil (150 mg, 30%, Rf: 0.26
in 2.5% CH3OH and 2.5% triethylamine in dichloromethane).
Cleavage of the ethylene bridges was observed during hydrogenation
and produced large amounts of byproduct. 1H NMR (300 MHz,
CDCl3): δ 3.28 (s, 2H, (CH3)3CO−CO−CH2−N′), 3.25 (s, 4H,
(CH3)3CO−CO−CH2−N/N″), 2.77−2.73 (m, 4H, ethylene-H),
2.62−2.59 (m, 4H, ethylene-H), 2.14 (s, 2H, −NH−), 1.40 (s, 18H,
(CH3)3CO−CO−CH2−N/N″), 1.39 (s, 9H, (CH3)3CO−CO−CH2−
H2dedpa. The hexadentate chelator H2dedpa was synthesized
according to the literature,33,35,36 with a modified purification step
performed by reverse-phase HPLC (gradient: A: 0.1% TFA, B:
CH3CN; 0 to 100% B linear gradient 25 min. tR = broad, 9.7−11 min),
followed by a second HPLC purification with a modified gradient (A:
distilled deionized water, B: CH3CN; 0 to 100% B linear gradient 25
min. tR = broad, 8.5−10 min).
[In(dedpa)]Cl. A portion of H2dedpa (10 mg, 0.020 mmol) and
In(ClO4)3·8H2O (13 mg, 0.023 mmol, 1.2 equiv) were dissolved in
HCl (aq) (1 mL, 0.1 M) in a 1 dram screw-cap vial. The pH was
adjusted to ∼4−4.5 with NaOH (aq) (0.1 M) while stirring. The
K
dx.doi.org/10.1021/ja3024725 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
reaction solution was stirred at 60 °C for 16 h, then evaporated to
dryness to afford [In(dedpa)]Cl as a white solid. 1H NMR (600 MHz,
DMSO-d6): δ 8.19 (t, 2H, pyr-H), 8.07 (d, 2H, pyr-H), 7.71 (d, 2H,
pyr-H), 4.40 (s, 2H, −NH−), 4.27/4.26 (two overlapping d, 2H, Pyr-
of In3+aq ion and the indium-chloride stability constants included in the
calculations were taken from Baes and Mesmer.66 All values and errors
represent the average of at least three independent experiments.
Molecular Modeling. Calculations were performed using the
Gaussian 0967 and GaussView packages. Molecular geometries and
electron densities were obtained from density functional theory
calculations, with the B3LYP functional employing the 6-31+G(d,p)
basis set for first- and second-row elements, and the ECP basis set,
LANL2DZ, was employed for indium.68,69 Solvent (water) effects were
described through a continuum approach by means of the IEF PCM as
implemented in G09. The electrostatic potential was mapped onto the
calculated electron density surface.
2
CH2−N, J = 16.6/16.3 Hz), 3.90/3.89 (two overlapping d, 2H, Pyr-
2
1
CH2−N, J = 16.6 Hz), 2.63−2.56 (m, 4H, ethylene-H). H NMR
(400 MHz, D2O): δ 8.28 (t, 2H, pyr-H), 8.20 (d, 2H, pyr-H), 7.79 (d,
2H, pyr-H), 4.58 (d, 2H, Pyr-CH2−N, 2J = 17.3 Hz), 4.13 (d, 2H, Pyr-
2
CH2−N, J = 17.4 Hz), 2.96−2.81 (m, 4H, ethylene-H). 13C NMR
(300 MHz, DMSO-d6): δ 163.74, 151.99, 146.94, 141.34, 124.62,
121.82, 49.48, 44.92. HR-ESI-MS calcd for [C16H16115InN4O4]+:
443.0210; found [M]+: 443.0216.
111In Radiolabeling Studies. The chelators H4octapa, H5decapa,
H2dedpa, DTPA, and DOTA were made up as stock solutions (1 mg/
mL, ∼10−3 M) in deionized water. An aliquot of each chelator stock
solution was transferred to screw-cap mass spectrometry vials and
made up to 1 mL with pH 5.5 NaOAc (10 mM) buffer, to a final
chelator concentration of ∼365 μM for each sample. A ∼10 μL aliquot
of the 111InCl3 stock solution (∼1 mCi for labeling studies and ∼5−7
mCi for mouse serum competitions) was transferred into the vials
containing each chelator, allowed to radiolabel at ambient temperature
for 10 min, and then analyzed by RP-HPLC to confirm radiolabeling
and calculate yields. [111In(DOTA)]− was heated at 80 °C for 20 min
in a microwave reactor to achieve quantitative radiolabeling yields.
Areas under the peaks observed in the HPLC radiotrace were
integrated to determine radiolabeling yields. The highest specific
activity of 2.3 mCi/nmol obtained for H4octapa was the result of
labeling a 990 μL solution of 3.65 × 10−7 M chelate in NaOAc buffer
(pH 5.3, 10 mM) with 10 μL of 111In3+ in a 0.05 M HCl solution (1.02
mCi) for 10 min at ambient temperature in 97.5% radiolabeling yield.
Elution conditions used for RP-HPLC analysis were gradient: A: 10
mM NaOAc buffer pH 4.5, B: CH3CN; 0 to 100% B linear gradient 20
min. The radiometal complex [111In(dedpa)]+ used a modified HPLC
gradient of A: 10 mM NaOAc buffer pH 4.5, B: CH3CN; 0 to 5% B
linear gradient 20 min. [111In(dedpa)]+ (tR = 5.9 min), [111In-
(octapa)]− (tR = 4.7 min), [111In(decapa)]2− (tR = 5.4 min {5%}, 7.7
min {95%}), [111In(DTPA)]2− (tR = 6.5 min), and [111In(DOTA)]−
(tR = 3.5 min) were all formed in >99% radiochemical yields.
Overlayed HPLC radiotraces are shown in Figure S27, SI, and some
original radiotraces are shown in Figures S19−S22, SI.
Solution Thermodynamics. The experimental procedures and
details of the apparatus closely followed those of a previous study for
H2dedpa with Ga3+.33 As a result of the strength of the binding of the
In3+ complex [111In(octapa)]−, the complex formation constant with
this ligand could not be determined directly and the ligand−ligand
competition method using the known competitor Na2H2EDTA was
used. For H5decapa, the ligand−ligand competition method was not
required owing to the presence of a stable MHL species, and instead it
was titrated directly with In3+. 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 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.
Indium ion solutions were prepared by dilution of the appropriate
atomic absorption standard (AAS) solution. The exact amount of acid
present in the indium standard was determined by titration of an
equimolar solution of In3+ and Na2H2EDTA. The amount of acid
present was determined by Gran’s method.63 Calibration of the
electrode was performed prior to each measurement by titrating a
known amount of HCl with 0.1 M NaOH. Calibration data were
analyzed by standard computer treatment provided within the
program MacCalib64 to obtain the calibration parameters E0.
Equilibration times for titrations were 10 min for pKa titrations and
15 min for metal complex titrations. Ligand and metal concentrations
were in the range of 0.75−1.0 mM for potentiometric titrations. The
data were treated by the program Hyperquad2008.65 The four
successive proton dissociation constants corresponding to hydrolysis
Mouse Serum Stability Data. The compounds [111In(octapa)]−,
[
111In(decapa)]2−, [111In(DOTA)]−, [111In(DTPA)]2−, and [111In-
(dedpa)]+ were prepared with the radiolabeling protocol as described
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 μL
mouse serum, 500 μL of 111In-complex (10 mM NaOAc buffer, pH
5.5), and 250 μL phosphate buffered saline (PBS) and were left to sit
at ambient temperature. After 1 h, half of the mouse serum
competition mixture (750 μL) 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 a 111In
waste container, and then the PD-10 column was eluted with 3.5 mL
PBS and collected into another sterile vial. The 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 2 represent the percentage of 111In that was retained on the
PD-10 column and therefore still chelate-bound.
Biodistribution Data. The protocol used in these animal studies
was approved by the Institutional Animal Care Committee (IACC) of
the University of British Columbia (Protocol # A10-0171) and was
performed in accordance with the Canadian Council on Animal Care
Guidelines. A total of 16 female ICR mice (20−25 g, 6−8 weeks) were
used for the biodistribution study of each of the three radiometal
complexes. [111In(octapa)]−, [111In(DOTA)]−, and [111In(decapa)]2−
were prepared as described above and then diluted in phosphate-
buffered saline to a concentration of 100 μCi/mL. Each mouse was
intravenously injected through the tail vein with ∼10 μCi (100 μL) of
the 111In complex and then sacrificed by CO2 inhalation 15 min, 1 h, 4
h, or 24 h after injection (n = 4 at each time point). Blood (500−700
μL) was collected by cardiac puncture with a 25 G needle and placed
into an appropriate microtainer tube for scintillation counting. Urine
was collected from the bladder after sacrifice and counted. Tissues
collected included spleen, kidney, intestine, liver, heart, lung, brain,
muscle, and femur. Tissues were weighed and counted with a Capintec
CRC 15R well counter, and the counts were decay corrected from the
first 15 min time point and then converted to the percentage of
injected dose (% ID) per gram of organ tissue (% ID/g).
ASSOCIATED CONTENT
■
S
* Supporting Information
Data and figures from potentiometric titrations, mouse serum
stability assays, mouse biodistribution studies, original HPLC
1
radio-traces, and H/13C NMR spectra of several compounds
and metal−chelate complexes. This material is available free of
charge via the Internet at http://pubs.acs.org.
L
dx.doi.org/10.1021/ja3024725 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
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(22) Sprague, J. E.; Peng, Y.; Fiamengo, A. L.; Woodin, K. S.;
Southwick, E. A.; Weisman, G. R.; Wong, E. H.; Golen, J. A.;
Rheingold, A. L.; Anderson, C. J. J. Med. Chem. 2007, 50, 2527−2535.
(23) Garrison, J. C.; Rold, T. L.; Sieckman, G. L.; Figueroa, S. D.;
Volkert, W. A.; Jurisson, S. S.; Hoffman, T. J. J. Nucl. Med. 2007, 48,
1327−1337.
AUTHOR INFORMATION
■
Corresponding Author
adam@triumf.ca; orvig@chem.ubc.ca
Notes
The authors declare no competing financial interest.
(24) Nayak, T. K.; Garmestani, K.; Baidoo, K. E.; Milenic, D. E.;
Brechbiel, M. W. J. Nucl. Med. 2010, 51, 942−950.
ACKNOWLEDGMENTS
■
(25) Schneider, D. W.; Heitner, T.; Alicke, B.; Light, D. R.; McLean,
K.; Satozawa, N.; Parry, G.; Yoo, J.; Lewis, J. S.; Parry, R. J. Nucl. Med.
2009, 50, 435−443.
We acknowledge Nordion (Canada) and the Natural Sciences
and Engineering Research Council (NSERC) of Canada for
grant support and for a CGS-M/CGS-D fellowship (E.W.P.),
and Dr. Dawn Waterhouse from the BC Cancer Agency for
performing the biodistribution experiments. C.O. acknowledges
the Canada Council for the Arts for a Killam Research
Fellowship (2011-2013).
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