Synthesis and Evaluation of Bifunctional [2.2.2]-Cryptands for Nuclear Medicine Applications

Article abstract of DOI:10.1021/acs.inorgchem.1c01274

For the first time, synthesis of bifunctional [2.2.2]-cryptands (CRYPT) and demonstration of radiolabeling with lead(II) (Pb2+) isotopes are disclosed herein. The synthesis is convenient and high-yielding and gives access to three distinct bifunctional handles (azide (-N3), isothiocyanate (-NCS), and tetrazine (-Tz)) that can enable the construction of radioimmunoconjugates for targeted and pretargeted therapy. Proof-of-principle CRYPT radiolabeling was successful with lead-203 ([203Pb]Pb2+) and demonstrated complexation efficiency superior to that of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and efficiency comparable to that of the current industry standard TCMC (1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane). In vitro human serum stability assays demonstrated excellent [203Pb]Pb-CRYPT stability over 72 h (91.7 ± 0.56%; n = 3). [203Pb]Pb-CRYPT-radioimmunoconjugates were synthesized from the corresponding CRYPT-immunoconjugate or by conjugating [203Pb]Pb-Tz-CRYPT to transcyclooctene modified trastuzumab (TCO-trastuzumab) via the inverse electron-demand Diels-Alder (IEEDA) reaction. This investigation reveals the potential for CRYPT ligands to become new industry standards for therapeutic and diagnostic radiometals in radiopharmaceutical elaboration.

Full text of DOI:10.1021/acs.inorgchem.1c01274

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Synthesis and Evaluation of Bifunctional [2.2.2]-Cryptands for
Nuclear Medicine Applications
Anthony W. McDonagh, Brooke L. McNeil, Brian O. Patrick, and Caterina F. Ramogida*
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ABSTRACT: For the first time, synthesis of bifunctional [2.2.2]-
cryptands (CRYPT) and demonstration of radiolabeling with
lead(II) (Pb2+) isotopes are disclosed herein. The synthesis is
convenient and high-yielding and gives access to three distinct
bifunctional handles (azide (−N3), isothiocyanate (−NCS), and
tetrazine (−Tz)) that can enable the construction of radio-
immunoconjugates for targeted and pretargeted therapy. Proof-of-
principle CRYPT radiolabeling was successful with lead-203
([203Pb]Pb2+) and demonstrated complexation efficiency superior
to that of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid) and efficiency comparable to that of the current industry standard TCMC (1,4,7,10-tetraaza-1,4,7,10-tetra-(2-
carbamoylmethyl)-cyclododecane). In vitro human serum stability assays demonstrated excellent [203Pb]Pb-CRYPT stability over 72
h (91.7
0.56%; n = 3). [203Pb]Pb-CRYPT-radioimmunoconjugates were synthesized from the corresponding CRYPT-
immunoconjugate or by conjugating [203Pb]Pb-Tz-CRYPT to transcyclooctene modified trastuzumab (TCO-trastuzumab) via the
inverse electron-demand Diels−Alder (IEEDA) reaction. This investigation reveals the potential for CRYPT ligands to become new
industry standards for therapeutic and diagnostic radiometals in radiopharmaceutical elaboration.
covalently bound to the linker or targeting vector. Designing
BFCs is a key component for a fully functioning inorganic
radiopharmaceutical and must meet specific requirements to
be optimal. These include being synthetically accessible, being
able to bind the radiometal under radiopharmaceutical
conditions (mild temperatures, submicromolar ligand concen-
trations, fast reaction times), and forming kinetically inert
complexes which are stable to transchelation in vivo. This is an
active area of research with several BFCs being developed over
the years.4,5 For example, Figure 1 illustrates a handful of
popular BFCs (1−4) used in nuclear medicine with different
bifunctional arms and coordinating environments. There is,
however, an opportunity for developing new ligands, and their
bifunctional variants, for radiopharmaceutical purposes with
the potential to surpass, in terms of function and application,
those that are currently used.
INTRODUCTION
■
Over the last 20 years, radiopharmaceutical therapy (RPT) has
emerged as an alternative for the treatment and diagnosis of
cancer. Unlike traditional radiotherapy, where radiation beams
are administered ex vivo, RPT involves delivering radiation
payloads directly and specifically to cancer cells in vivo.1 This
method has several advantages over existing therapeutic
strategies, including attenuated radiation dose, reduced side
effects, and rapid drug response, and enables in vivo
visualization through positron emission tomography (PET)
or single-photon emission computed tomography (SPECT).1
Metallic radioisotopes (also termed radiometals) possess
vast diversity in terms of their physical properties (e.g.,
radiological half-life and decay emissions), making them ideal
for incorporation into radiopharmaceuticals. A metal-based
radiopharmaceutical can generally be broken down into four
components consisting of the radiometal, bifunctional chelator
(BFC), linker, and biomolecule/targeting vector.2 The decay
properties of the radiometal provide the diagnosis/treatment,
while the biomolecule/targeting vector ensures accumulation
of the drug at the point of infection. These two components
are then combined through the BFC and linker. Several
radiopharmaceuticals bearing this blueprint are currently in
clinical trials,1 and recently their potential was recognized with
the FDA approval of lutetium-177 ([177Lu]) Lu-DOTATATE,
a radioactive drug used for the treatment of neuroendocrine
tumors.3 BFCs contain a multidentate ligand, which
coordinates the radionuclide, and a reactive arm that can be
[2.2.2]-cryptand (Kryptofix 222) 5 is a three-dimensonal
N2O6 donating “cage like” structure synthesized by Lehn in the
late 1960s.6,7 Since its discovery, the ligand has demonstrated a
myriad of applications in chromatography,8,9 organic syn-
Received: May 4, 2021
Published: June 23, 2021
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Figure 1. (left) Structures of popular bifunctional chelators. (right) Illustrating the concept for bifunctional [2.2.2]-cryptands developed in this
work in comparison to the original structure.
thesis,10 crystallography,11−13 electrochemistry,14,15 chemical
RESULTS AND DISCUSSION
■
sensing,16 and magnetic resonance imaging.17−19 Surprisingly,
Synthesis and Characterization. The synthesis began by
transforming phenol32 9 into the required bifunctional arm 14
to the best of our knowledge, [2.2.2]-cryptand has never been
radiolabeled and developed into a BFC. Its only application in
radiochemistry is as a phase-transfer catalyst when synthesizing
the PET imaging agent [18F]fluoro-2-deoxy-2-D-glucose ([18F]-
FDG).20 Some N6 donating cryptands, such as DiAmSar and
its subsequent bifunctional analogues (e.g., AmBaSAr 2, Figure
1), have been developed for copper-64 and -67 (64Cu/67Cu)
radiolabeling and form remarkably kinetically inert Cu
complexes.21−24 However, synthesizing the bifunctional
analogues can be challenging, and its small cavity size limits
its application to only small radionuclides. It is therefore worth
investigating if the [2.2.2]-cryptand could be made bifunctional
and radiolabeled for nuclear medicine applications. The
pioneering work for [2.2.2]-cryptand-metal complexes has
primarily focused on alkali metal cations. However, very
recently the ligand has been demonstrated to coordinate
lanthanides, actinides, and other large metal cations analogous
to radiometals. These complexes exhibit extraordinary
thermodynamic and kinetic stability and are highly resistant
toward transchelation.12,25−28 We rationalized that the trend
may carry over to radiometals and potentially lead to new
ligands that bind radiometals under radiopharmaceutical
conditions (vide supra) to form exceptionally thermodynami-
cally stable and kinetically inert complexes in vivo. The main
challenge would be making the cryptand bifunctional and
employing a reproducible synthetic route that could be easily
implemented. The original structure was therefore redesigned
to contain an aromatic functionality bearing a novel bifunc-
tional arm (Figure 1, structures 6−8). Disclosed herein are the
results of this investigation which illustrate the first synthesis of
three bifunctional [2.2.2]-cryptands for potential use in
imaging, targeted or pretargeted RPT. Their bifunctional
utility was demonstrated, and proof of principle radiolabeling
was undertaken and optimized with γ-emitter lead-203 (203Pb,
t1/2 = 51.9 h). Finally, the first CRYPT-radioimmunoconju-
gates was synthesized by coupling to the HER2/neu-targeting
monoclonal antibody (mAb) trastuzumab (Herceptin).29−31
(Scheme 1). Alkylation of 9 with methyl 4-bromobutyrate gave
Scheme 1. Synthesis of Bifunctional Arm 14
intermediate 10, which was subsequently treated with aqueous
trifluoroacetic acid (TFA) to give 11 in excellent yield (66%
for two steps). The alkylation deprotection protocol was then
repeated by treating 11 with tert-butyl bromoacetate under
basic conditions to give 12; the tert-butyl esters were then
removed with TFA to give diacid 13. Treating 13 with oxalyl
chloride then gave 14 in quantitative yield (88% for three
steps). With the diacid chloride intermediate 14 in hand,
amide coupling to 4,13-diaza-18-crown-6 15 was undertaken
(Scheme 2). High dilution in toluene for this transformation is
necessary to obtain diamide 16 in good yield (67%).19
Following this, chemoselective reduction of the amide over the
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Scheme 2. Synthesis of the Bifunctional [2.2.2]-Cryptands 6, 7, and 8
ester in 16 was attempted with 9-BBN.33 The initial synthetic
Our attention then turned to choosing a suitable radiometal
for the cryptand. Of the number of radioisotopes available, and
with the cavity size of [2.2.2]-cryptand being 2.8 Å, we
hypothesized that the diagnostic radiometal 203PbII (t1/2 = 52 h,
ionic radius = 1.29 Å for coordination number (CN) = 8)40
would fit within the cryptand cavity; 203Pb is also readily
available via irradiation of thallium on a medical cyclotron and
produced regularly at our facility. Moreover, the cryptand
provides potentially 8-donor atoms of borderline to moderate
basicity, two 3° amines and six ether oxygens, which we
believed would satisfy the coordination chemistry of Pb(II)
which is characterized as a borderline Lewis acid according to
Pearson’s hard soft acid base theory. Prior to radiolabeling
studies, the nonradioactive natPbII-cryptand complex was
prepared and characterized. Gratifyingly, upon treating 17
with lead(II) perchlorate (Pb(ClO4)2), the corresponding
cryptand lead complex [Pb(17)ClO4][ClO4] was isolated in
design for a bifunctional cryptand was to perform a
chemoselective reduction of the amide, saponify the ester to
the corresponding acid, and then covert into a succinimidyl
ester. However, the transformation with 9-BBN was
unsuccessful, giving poor yield of the desired product and
complex reaction mixtures. 16 was therefore treated with
borane-tetrahydrofuran (BH3·THF) to simultaneously reduce
the diamide and the methyl ester to give cryptand 17 in good
yield (74%).19 Oxidation of the primary alcohol on 17 to the
corresponding carboxylic acid was unsuccessful with TEMPO/
BAIB,34 Jones Reagent,35 and with pyridinium dichromate in
DMF.36
Activation of the alcohol with triphosgene and disuccini-
midyl carbonate (DSC) was also attempted. Both the
chloroformate and succinimidyl carbamate could be detected
and trapped with a primary amine to give the corresponding
carbamate. However, purification of the products was too
difficult and not reproducible. It was therefore decided to
convert the alcohol to an azide by treating 17 with methane
sulfonyl chloride to give the corresponding mesylate, which
was immediately treated with sodium azide (NaN3) in DMF at
80 °C to give 6 in excellent yield (79% for two steps).37 It is
worth noting that the azide-cryptand (N3-CRYPT) 6 is
bifunctional and can be used for strained promoted azide−
alkyne cycloaddition (SPAAC) to synthesize bioconjugates.38
Following this, 6 was transformed to primary amine 18 via
palladium-catalyzed hydrogenation in almost quantitative yield
(98%). To complete the synthesis, the amino-cryptand was
then converted into two more novel bifunctional cryptands. 18
was treated with carbon disulfide, to give an intermediate
dithiocarbamate, which was transformed in situ with acetyl
chloride to the corresponding isothiocyanate-cryptand (NCS-
CRYPT) 7 in moderate yield (43%). Alternatively, 18 can be
conjugated with tetrazine-NHS ester39 to give tetrazine-
cryptand (Tz-CRYPT) 8 in good yield (67%). In general,
starting from the diacid chloride intermediate 14, each of the
bifunctional cryptands 6, 7, or 8 can be prepared in under 5
days.
1
quantitative yield (Scheme 3). The H NMR spectrum of
Scheme 3. Synthesis of Nonradioactive Lead(II) Complex
[Pb(17)ClO4]ClO4 from Pb(ClO4)2 with 1H NMR Labeling
Assignments
[Pb(17)ClO4][ClO4] (Figure 2) showed clear downfield shifts
for each CH2 within the cryptand core compared to the
uncoordinated ligand 17, thereby suggesting that each
heteroatom was interacting to form an 8-coordionate solution
structure. There was no dramatic chemical shift change with
the side arm methylene protons, indicating that this moiety
1
played no part in forming the complex. The H NMR also
confirms that only one highly symmetric isomer is present in
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Figure 2. 1H NMR (500 MHz) spectra in CD3OD-d4 at ambient temperature of ligand 17 (top) and the corresponding lead complex
[Pb(17)ClO4]ClO4 (bottom).
solution, an advantage because complexes that form a
minimum number of isomers are more stable in vivo, which
is a key component for a successful radiopharmaceutical.41,42
Next was to gain insight into the solid-state structure;
however, crystallization attempts of [Pb(17)ClO4][ClO4] were
unsuccessful. It was rationalized the side arm might be
influencing crystal growth. Instead, the core [2.2.2]-cryptand
structure bearing no functional side arm (19) was synthesized
via a novel route similar to 17 (see the Supporting
Information) and reacted with Pb(ClO4)2 to give [Pb(19)-
ClO4][ClO4] from which X-ray quality crystals were obtained
through vapor diffusion (Figure 3). The X-ray structure shows
PbII residing in the center of the cryptand bound by the eight
[ClO4] (N−Pb (2.8 Å), aliphatic O−Pb (∼2.6 Å), aromatic
O−Pb (2.9, 3.0 Å), and perchlorate O−Pb (2.9 Å)) are similar
to [U(crypt)I2][I],25 [Nd(crypt)OTf2][OTf],27 and [Eu-
(crypt)Cl][Cl]19 metal complexes. The ionic radius for PbII
(1.4 Å for coordination number (CN) = 10) is slightly larger
than but comparable to that for Eu2+, La3+, U3+, and Nd3+
when coordinated to cryptands (Table 1).
Prior to radiolabeling studies, the bifunctional application of
cryptands NCS-CRYPT (7) and Tz-CRYPT (8) was
demonstrated. The isothiocyanate (−NSC) and tetrazine
(−Tz) functionalities can be employed in targeted or
pretargeted radiopharmaceutical formulation, respectively.
1
Table 1. Comparing the Bond Lengths of
N2O6 donor atoms (suggested by H NMR) as well as one
perchlorate counterion through both O atoms to form a 10-
coordinate complex. The bond lengths for [Pb(19)ClO4]-
[Pb(19)ClO4][ClO4] and Metal Atomic Radii with
a
Literature Metal−[2.2.2]-Cryptand Complexes
bond length (Å)
O−M
O−M
M−Ionic
b
M−crypt complex
N−M aliphatic aromatic radius (CN)
[Pb(19)ClO4][ClO4]
[Eu(crypt)Cl][Cl]19
[La(crypt)Cl2][Cl]25
[U(crypt)I2][I]25
2.9
[Nd(crypt)OTf2][OTf]27 2.8
2.8
2.8
2.9
2.8,
2.6
2.7
2.7
2.57,
2.70
2.55, 2.6
2.9, 3.0
2.7, 2.8
NA
1.4 (10)
1.3 (9)
1.27 (10)
c
NA
ND (10)
d
NA
ND (10)
a
NA (not applicable) refers to structures that do not contain an
aromatic ring O−M bond length. CN, coordination number.
b
c
Reported metal ionic radii taken from.40 ND = not determined
(no reported value for U(III) ion [CN = 10]; ionic radius for U(III)
d
ion [CN = 6] reported as 1.03 Å). ND = not determined (no
reported value for Nd(III) ion [CN = 10]; ionic radii for Nd(III)
[CN = 9, 12] reported as 1.16 and 1.27 Å, respectively).
Figure 3. Solid-state X-ray crystal structure of [Pb(19)ClO4][ClO4]
synthesized from the core nonbifunctional cryptand 19.
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Scheme 4. Illustrating the Synthesis of Trastuzumab-Cryptand Immunoconjugates 20 and 21, from NCS-CRYPT (7) and
a
Trastuzumab (top) or from TCO-Trastuzumab and Tz-CRYPT (8) (bottom)
a
The average number of ligands per antibody was determined by MALDI-TOF MS/MS to be 2.1 for 20 and 4.3 for 21.
Generally, in targeted imaging or therapy, an immunoconju-
gate is synthesized from reacting the antibody lysine residues
with the bifunctional arm (isothiocyanate, succinimide, azide,
etc.) of a BFC, then labeled with a multiday half-life
radionuclide, and injected in vivo to accumulate at a tumor.
In pretargeted therapy, a trans-cyclooctene (TCO) modified
antibody, which can only react with a tetrazine residue via the
inverse electron-demand Diels−Alder (IEDDA) reaction, is
administered in vivo. The antibody slowly accumulates at the
tumor and clears from the blood. A radiolabeled chelator
bearing a tetrazine is then injected and rapidly conjugates to
only the TCO-antibody in vivo.43,44 Pretargeted therapy is
becoming popular over targeted therapy because short-lived
radionuclides can be utilized and the overall radiation dose is
significantly reduced. The synthesized bifunctional cryptands
therefore have potential to be used for targeted (N3-CRYPT,
NCS-CRYPT, and Tz-CRYPT) or pretargeted (Tz-CRYPT)
therapy. As a proof of principle, the conjugation of the
bifunctional cryptands 7 and 8 with the monoclonal antibody
trastuzumab was investigated (Scheme 4). For the NCS-
CRYPT (7), the conditions involved incubating the ligand
with the antibody in PBS buffer (pH 8.0−9.0) at 37 °C for 90
min.45 The conjugation with the Tz-CRYPT (8) was
preformed with TCO-trastuzumab (synthesized as per
previously published procedures)44 and incubated in PBS
buffer (pH 7.0) at 25 °C for 60 min.46 After purifying each
conjugation reaction via PD-10 size exclusion, both the
corresponding thiourea 20 and 1,2-dihydropyrazine 21
immunoconjugates were confirmed by MALDI-TOF MS/
MS. The average number of chelators per antibody were 2.1
for 20 and 4.3 for 21.
represents the first example of radiolabeling [2.2.2]-cryptands,
it was predicted optimization would be necessary to obtain
sufficient radiochemical yields (RCYs). OH-CRYPT (17) was
therefore chosen as a model substrate to investigate the initial
radiolabeling performance. Cryptands are reported to bind
50
alkali metal (Li+, Na+, K+)47−49 and ammonium (NH4 )
+
cations, and therefore, because radiolabeling reactions are
generally carried out in buffers containing these cations, they
may compete with the radiometal, especially at lower
concentrations. Keeping this in mind, 17 ([L] = 10−4 M)
was incubated with 203Pb (40−60 kBq)52 in either 1 or 0.1 M
ammonium acetate (NH4OAc, pH 7). Further, recent reports
of improved radiolabeling yields in organic solvents compared
to aqueous buffers motivated our investigation of 203Pb
labeling in methanol (MeOH), ethanol (EtOH), acetonitrile
(CH3CN), and dimethyl sulfoxide (DMSO).51 Nonisolated
RCYs were determined via radio-instant thin layer chromatog-
raphy (radio-iTLC) after 5 and 60 min at room temperature or
80 °C. Complexation was observed at ambient temperature for
each reaction with RCYs after 60 min of >99% (1 M NH4OAc,
pH 7), >99% (0.1 M NH4OAc, pH 7), 94.2 3.8% (MeOH),
94.6
0.9% (EtOH), 94.7
1.1% (CH3CN), and 30.2
2.4% (DMSO); n = 3 for each experiment. The reactions were
incomplete after 5 min, and elevated temperature (80 °C) had
no effect on rate or RCY. Because preliminary 203Pb labeling in
organic solvents did not yield quantitative labeling, further
experiments in MeOH, EtOH, CH3CN or DMSO were not
attempted. Concentration-dependent (10−4−10−7 M) radio-
labeling in 1 and 0.1 M NH4OAc (pH 7) was conducted. The
RCY in 1 M NH4OAc unfortunately decreased significantly at
10−5 M (25.7 4.6%; n = 3) and 10−6 M (3.8 1.3%; n = 3)
ligand concentration. However, the RCY with 0.1 M NH4OAc
at 10−5 M was >99%, n = 3 and 88.6% 6.0%, n = 3 for 10−6
Radiolabeling Experiments. With our novel bifunctional
[2.2.2]-cryptands and immunoconjugates in hand, radio-
labeling with [203Pb]Pb2+ was investigated. Because this
+
M and suggests the NH4 was competing with 203Pb at higher
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buffer concentrations. Using the optimized labeling conditions
(0.1 M NH4OAc, pH 7, room temperature, 60 min), a
comparative labeling investigation between CRYPT 17 and
commercial standards for 203Pb chelation, DOTA, and TCMC
(Figure 4), ([L] = 10−4−10−7 M) was conducted. All three
percentage of intact 203Pb was monitored by iTLC at 3 h, 24 h,
48 and 72 h. The complex remained >91% over the course of
the study, thereby demonstrating exceptional stability for the
radiolabeled cryptand.
Finally, to demonstrate the ability of the bifunctional
cryptands to form radioimmunoconjugates, the optimized
203Pb radiolabeling was performed with immunoconjugate 20
and Tz-CRYPT 8. As mentioned, targeted therapy involves a
radiolabeled immunoconjugate, whereas in pretargeting, a
tetrazine-bearing BFC is labeled and conjugated to a TCO-
modified antibody. 20 (50, 25, or 10 μg) was incubated with
203Pb (50 kBq) for 60 min at room temperature, and the
corresponding radioimmunoconjugate formation was con-
firmed via iTLC for each reaction with RCYs of 88.1
0.1%, 83.2
0.4%, and 77.7
0.6% (n = 3 for each
experiment), respectively. Further, Tz-CRYPT 8 was radio-
labeled with 203Pb (50 kBq) in quantitative yield. The
[
203Pb]Pb-Tz-CRYPT was observed to be stable in human
serum over 72 h (Table 2), and the IEDDA reaction was
performed by incubating the radioligand with TCO-trastuzu-
mab at different molar ratios (2:1, 4:1, and 10:1 Tz-CRYPT to
TCO-trastuzumab).46 After 60 min at room temperature each
reaction was purified via PD-10 size exclusion, and the
corresponding radioimmunoconjugate was confirmed by γ
spectroscopy of the final eluate to give radiolabeling yields of
51%, 60%, and 39%, respectively.
Figure 4. Radiochemical yields (RCYs) at various ligand concen-
trations for OH-CRYPT 17 (1 and 0.1 M NH4OAc, pH 7), TCMC
(0.1 M NH4OAc, pH 7), and DOTA (0.1 M NH4OAc, pH 7)
complexation with [203Pb]Pb(OAc)2 at 1 h and room temperature.
ligands quantitatively (RCY > 99%) complexed 203Pb at ligand
concentrations of 10−4 and 10−5 M. At 10−6 M the RCY for
CONCLUSION
■
In summary, we have disclosed the synthesis of novel
bifunctional [2.2.2]-cryptands and demonstrated their poten-
tial use in nuclear medicine for the first time. A novel
bifunctional arm was synthesized in five steps with a 58%
overall yield and conjugated to 4,13-diaza-18-crown-6. The
corresponding diamide intermediate was conveniently trans-
formed into three novel bifunctional cryptands bearing an
azide (N3-CRYPT), an isothiocyanate (NCS-CRYPT), or a
tetrazine (Tz-CRYPT) with 39%, 17%, and 26% overall yields,
respectively. Nonradioactive Pb-complexes of OH-CRYPT
(17) and the nonbifunctional analogue (CRYPT) were
prepared and characterized; 1H NMR suggested an 8-
coordinate solution structure, which forms a single isomer.
X-ray crystallography revealed a 10-coordinate solid-state
structure bound by the eight N2O6 donor atoms as well as
one perchlorate counterion. Furthermore, the bifunctional
application for NCS-CRYPT and Tz-CRYPT was demon-
strated by conjugating each BFC to the monoclonal antibody
trastuzumab. CRYPT-radiolabeling was successful with SPECT
imaging isotope 203Pb; the transformation then was optimized
to obtain high RCYs. Finally, the preclinical application was
demonstrated by synthesizing [203Pb]Pb-CRYPT radioimmu-
noconjugates for targeted or pretargeted therapy. The success
of this investigation highlights a potential application for
CRYPT ligands in nuclear medicine. Herein, we have shown
radiolabeling with 203Pb2+; however, there are a myriad of
radioisotopes available for testing (225Ac3+, 111In3+, 64Cu2+,
212Pb2+, etc.). CRYPT could find use for many radiometals and
lead to new radiopharmaceuticals to treat and diagnose
diseases. Further experimentation is now underway and will
be reported in due course.
DOTA dropped to 23.9
maintained near quantitative RCYs (98.4 1.2%, n = 3), and
the CRYPT dropped to 88.6 6.0%, n = 3. Then at 10−7
TCMC and DOTA were 5.0 2.4% and 1.5 0.6%,
respectively (each n = 3), and CRYPT was slightly higher at
13.6 3.0%, n = 3. These results suggest that, under these
7.3%, n = 3 while TCMC
M
conditions, the cryptand is superior to DOTA and comparable
to TCMC at complexing 203Pb. The kinetic inertness of the
resulting [203Pb]Pb-CRYPT complex was evaluated in an in
vitro human serum stability assay (Table 2). Radiopharmaceu-
Table 2. Human Serum Stability Challenge Data Performed
at 37 °C (n = 3), with Stability Shown as Percentage of
Intact 203Pb Complex
a
no.
1−8 h
24 h
48 h
72 h
1
2
3
4
93.0 1.0% (3 h) 92.0 1.5%
97.2 1.2% (1 h) 99.2 1.1%
97.1 0.7% (8 h) 97.3 0.6%
98.0 0.5% (8 h) 98.1 0.2%
94.0 1.0%
97.5 1.3%
98.1 0.2%
98.2 0.3%
91.6 0.6%
95.7 0.6%
97.4 0.5%
97.2 0.7%
a
1 [203Pb]Pb−OH-CRYPT, 2 [203Pb]Pb-Tz-CRYPT, 3 [203Pb]Pb-
DOTA,52 and 4 [203Pb]Pb-TCMC.52
ticals for targeted or pretargeted therapy are given by
intravenous (IV) injection, and because several endogenous
ligands, such as albumin and metallothionein, present in blood
serum can compete and displace chelator bound metal ions, it
is necessary to investigate if radiometal-chelate complexes can
withstand transchelation in vivo. Failure to do so would result
in unsuccessful delivery of the radiopharmaceuticals to the
desired target. [203Pb]Pb-CRYPT was incubated with human
serum under physiological conditions (37 °C, pH 7), and the
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c01274.
■
sı
Experimental procedures, H and 13C NMR spectra, X-
ray crystallography data, and iTLC and radio HPLC
chromatograms (PDF)
1
Accession Codes
CCDC 2080228 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Author
Caterina F. Ramogida − Department of Chemistry, Simon
Fraser University, Burnaby, BC V5A 1S6, Canada; Life
Sciences Division, TRIUMF, Vancouver, BC V6T 2A3,
Canada; orcid.org/0000-0003-4815-2647; Email: cfr@
sfu.ca
■
Authors
Anthony W. McDonagh − Department of Chemistry, Simon
Fraser University, Burnaby, BC V5A 1S6, Canada
Brooke L. McNeil − Department of Chemistry, Simon Fraser
University, Burnaby, BC V5A 1S6, Canada; Life Sciences
Division, TRIUMF, Vancouver, BC V6T 2A3, Canada
Brian O. Patrick − Department of Chemistry, University of
British Columbia, Vancouver, BC V6T 1Z1, Canada;
orcid.org/0000-0002-2921-8439
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.1c01274
Notes
The authors declare no competing financial interest.
(15) Zejli, H.; Hidalgo-Hidalgo de Cisneros, J. L.; Naranjo-
Rodriguez, I.; Elbouhouti, H.; Choukairi, M.; Bouchta, D.;
Temsamani, K. R. Electrochemical Analysis of Mercury Using a
Kryptofix Carbon-Paste Electrode. Anal. Lett. 2007, 40 (14), 2788−
2798.
(16) Li, J.; Yim, D.; Jang, W.-D.; Yoon, J. Recent Progress in the
Design and Applications of Fluorescence Probes Containing Crown
Ethers. Chem. Soc. Rev. 2017, 46 (9), 2437−2458.
(17) Garcia, J.; Neelavalli, J.; Haacke, E. M.; Allen, M. J. EuII-
Containing Cryptates as Contrast Agents for Ultra-High Field
Strength Magnetic Resonance Imaging. Chem. Commun. 2011, 47
(48), 12858.
ACKNOWLEDGMENTS
■
Funding for this work was provided by Natural Sciences and
Engineering Research Council (NSERC) of Canada Discovery
Grant (CFR, RGPIN-2019-07207) and a Social Sciences and
Humanities Research Council (SSHRC) of Canada New
Frontiers in Research Fund Exploration Grant (CFR, NFRFE-
2018-00499). TRIUMF receives funding via a contribution
agreement with the Natural Research Council of Canada. The
BC Cancer Agency (Dr. Julie Rousseau) is thanked for
contributing trastuzumab for this study.
(18) Garcia, J.; Allen, M. J. Interaction of Biphenyl-Functionalized
Eu2+-Containing Cryptate with Albumin: Implications to Contrast
Agents in Magnetic Resonance Imaging. Inorg. Chim. Acta 2012, 393,
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