Inorganic Chemistry
Article
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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Inorg. Chem. 2021, 60, 10030−10037