New cross-bridged cyclam derivative CB-TE1K1P, an improved bifunctional chelator for copper radionuclides

Article abstract of DOI:10.1039/c3cc45928d

A new cross-bridged cyclam chelator, CB-TE1K1P, was developed for copper-based radiopharmaceuticals, and this chelator can be labelled with ⁶⁴Cu under mild conditions in high specific activity. DBCO-PEG ₄-CB-TE1K1P was synthesized for conjugation to proteins, while Dde-CB-TE1K1P(^tBu₂)-OH was synthesized for solid-phase peptide synthesis. Examples of the conjugation chemistry, radiolabelling and serum stability of each are presented.

Full text of DOI:10.1039/c3cc45928d

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Carolyn J. Anderson et al.
New cross-bridged cyclam derivative CB-TE1K1P, an improved
bifunctional chelator for copper radionuclides

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New cross-bridged cyclam derivative CB-TE1K1P,
an improved bifunctional chelator for copper
radionuclides†
Cite this: Chem. Commun., 2014,
50, 43
Received 2nd August 2013,
Accepted 3rd October 2013
Dexing Zeng,a Qin Ouyang,b Zhengxin Cai,a Xiang-Qun Xieb and
Carolyn J. Anderson*a
DOI: 10.1039/c3cc45928d
www.rsc.org/chemcomm
A new cross-bridged cyclam chelator, CB-TE1K1P, was developed for
copper-based radiopharmaceuticals, and this chelator can be labelled
with 64Cu under mild conditions in high specific activity. DBCO–PEG4–
CB-TE1K1P was synthesized for conjugation to proteins, while Dde–
CB-TE1K1P(tBu2)–OH was synthesized for solid-phase peptide synthesis.
Examples of the conjugation chemistry, radiolabelling and serum stability
of each are presented.
Fig. 1 Chelators for copper radionuclides.
The copper radionuclides Cu-60, Cu-61, Cu-62, Cu-64 and Cu-67 have
applications in diagnostic imaging, radiotherapy, and theranostics.1–3
There are two principle oxidation states (I and II), and the coordina-
tion and redox chemistry of copper is well documented. Additionally,
the biochemistry and metabolism of copper are also well known,
since there are many mammalian and bacterial copper-containing
enzymes and proteins.4 The redox and kinetic properties of Cu(II)
present a challenge for the development of kinetically inert copper
complexes for diagnostic imaging and radiotherapy.
To prepare copper-based radiopharmaceuticals, the copper radio-
nuclides are incorporated into a biomolecule through a bifunctional
chelator (BFC). An optimal BFC should bind with copper rapidly under
mild conditions in high specific activity (SA). The resulting radiometal
complex should exhibit high thermodynamic stability,5,6 and more
importantly, also sufficient stability in vivo to withstand a challenge from
copper-containing proteins, such as ceruloplasmin and metallothio-
nein.7 Considerable efforts have been focused on the development of
novel BFCs for copper radionuclides, and a number of chelators, such as
DOTA, TETA, NOTA, TE2A, CB-TE2A and their analogues, have been
intensively studied as BFCs for this purpose (Fig. 1).8–12
temperature-sensitive molecules, such as proteins and heat-sensitive
peptides. Some of the newly developed chelators (Fig. S1, ESI†), such
as p-SCN-Bn-PCTA, p-SCN-Bn-Oxo,13 SarAr,14 CB-TE2A,15 were labelled
with Cu-64 under mild conditions in high specific activity; however,
none of these chelators demonstrated improved in vivo behavior
compared to CB-TE2A. Furthermore, these chelators cannot be directly
used as N-protected amino acid surrogates for SPPS (solid phase
peptide synthesis), and usually they are conjugated to peptides
through either an N-terminus or primary amine in the side chain.
We recently reported on CB-TE1A1P (Fig. 1), which can be
labelled at room temperature in high SA. The resulting 64Cu-
complex exhibited comparable or even better in vivo stability with
more rapid clearance through blood, kidneys and liver compared to
64Cu labelled NOTA, Diamsar and CB-TE2A.10 CB-TE1A1P can be
conjugated to peptides using a primary amine; however, the conver-
sion of the carboxylate group to an amide linkage potentially
decreases its in vivo stability. Therefore, an improved chelator that
can be rapidly labelled under mild conditions (e.g. short labelling
times at physiological temperature), and also maintains high stabi-
lity of the cross-bridge cyclam, would be highly attractive for the
design of copper-based radiopharmaceuticals.
Among these BFCs, CB-TE2A demonstrated excellent kinetic inert-
ness and in vivo stability, and has been recognized as one of the ‘‘gold
standards’’ in recent years. However, harsh radiolabelling conditions
are required (95 1C for 1 h), which hinder applications with
CB-TE1K1P contains a carboxylate and a methanephosphonate
pendant group for ease of labelling at room temperature with
maintained in vivo stability. Furthermore, in order to facilitate its
conjugation with biomolecules, two derivatives (7 and 9) were
prepared for facile conjugation to either proteins or peptides.
As illustrated in Scheme 1, the a-amino group in H-Lys(Cbz)-OH
a Department of Radiology, University of Pittsburgh, 100 Technology Drive,
Suite 452F, Pittsburgh, PA 15219, USA. E-mail: andersoncj@upmc.edu;
Fax: +1-412-624-2598; Tel: +1-412-624-6887
b Department of Pharmaceutical Sciences, University of Pittsburgh,
3501 Terrace Street, Pittsburgh, PA 15213, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc45928d was converted to the corresponding bromide, and 2 was obtained after
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ratio in the CB-TE1A1P–cetuximab conjugate is likely due to the poor
efficiency of the NHS-aided coupling reaction between CB-TE1A1P
and the primary amines of the antibody, possibly due to the effect of
the methanephosphonate group in CB-TE1A1P.17 Inspired by previous
reports on the conjugation of BFCs to nanoparticles via metal-free
click chemistry (also called strain-promoted alkyne–azide cycloaddi-
tion, SPAAC),18,19 we designed and synthesized 7 for conjugation to
azido functionalized cetuximab (Scheme 2a). The number of chelators
per antibody can be easily modified in the cetuximab azidolation step
by adjusting the amount of azide-NHS ester. The cetuximab conjugate
with B9 CB-TE1K1P chelators showed high binding affinity to the
EGFR receptor (Kd = 1.4 Æ 0.4 nM) and was used for further
evaluation. Two other cetuximab conjugates were also synthesized,
including CB-TE1A1P-click-cetuximab prepared using DBCO–CB-
TE1A1P, and non-click CB-TE1A1P–cetuximab prepared via a standard
Scheme 1 Synthesis of CB-TE1K1P and its two derivatives.
silica gel purification in 60.5% isolation yield. In the second step, the peptide coupling reaction. A comparison of the 64Cu–CB-TE1K1P–
carboxylic acid group of 2 was protected as a benzyl ester by coupling
cetuximab with other 64Cu-labeled conjugates is summarized in Fig. 2.
64Cu-labeled CB-TE1K1P-click-cetuximab with 9 chelates per anti-
with benzyl alcohol. The resulting compound 3 underwent mono-N-
alkylation of cross-bridged cyclam, yielding pure 4 after removing excess body had a specific activity of 685 kBq mgÀ1 after incubation at 37 1C
cross-bridged cyclam. Compound 4 was then converted to the mono-di- for 30 min (or 40 1C for 25 min). In contrast, after incubation at 40 1C
tert-butyl-phosphonate intermediate 5 via three-component Kabachnik– for 2 h, the 64Cu-labeled CB-TE1A1P-click-cetuximab with the same
Fields reaction. Intermediate 5 was hydrolyzed using 6 M HCl by number of chelates per antibody had a SA of 334 kBq mgÀ1, which
refluxing overnight and gave the product CB-TE1K1P (6) after purifica- may be attributed to the loss of the carboxylate group that facilitates
tion by ion exchange chromatography. 6 was functionalized with DBCO the copper ion complexation. The SA of 64Cu-labelled NHS conju-
(dibenzo-cyclooctyne) by forming a peptide bond, and after HPLC
purification yielded pure DBCO–PEG4–CB-TE1K1P (7) for conjugation
gated CB-TE1A1P–cetuximab with 2 chelates per antibody was only
111 kBq mgÀ1 when labelled for 2 h at 40 1C. These data demonstrate
to biomolecules via metal-free click chemistry. Compound 5 was readily that CB-TE1K1P is rapidly labelled with Cu-64 under mild conditions
converted to compound 8 after stirring with Pd/C under H2, and then in high SA (see Fig. 2a for details). Furthermore, both the
crude 8 was treated with DDe and yielded 9 that was purified by silica resulting 64Cu–CB-TE1K1P-click-cetuximab and 64Cu–CB-TE1A1P-
gel chromatography.
click-cetuximab exhibited good serum stability (Fig. 2b).
In order to demonstrate the utility of CB-TE1K1P, we prepared
Besides macromolecules, we also explored the application of CB-
two bioconjugates and labelled them with 64Cu. Compound 7 was TE1K1P in preparing 64Cu-labelled peptides. We designed and synthe-
sized Dde–CB-TE1K1P(tBu2)–OH (9) that is suitable for SPPS. Dde–CB-
conjugated to the anti-EGFR monoclonal antibody, cetuximab.
Compound 9 was attached to the cFlFlF peptide, which binds the
TE1K1P(tBu2)–OH was first loaded onto a Wang resin by forming an
N-formyl peptide receptor 1 (FPR-1).
ester bond, and then the primary amine in CB-TE1K1P was released
The conjugation and radiolabelling of CB-TE1K1P–PEG4-click- after deprotection with 4% hydrazine. The resulting resin pre-loaded
cetuximab were chosen since there have been no successful studies with CB-TE1K1P can be readily used for preparation of any peptide
of 64Cu-labelling of a cross-bridged macrocycle–antibody conjugate. using standard Fmoc chemistry based SPPS, and then the CB-TE1K1P
This was compared to direct conjugation of CB-TE1A1P with cetux- attached peptide can be subsequently cleaved using trifluoroacetic
imab, which resulted in an average of 2 chelators per antibody acid (Scheme 2b). By using this strategy, a neutrophil-targeted
measured using a published method.16 The low chelator/antibody
cFlFlF–PEG4–CB-TE1K1P conjugate was successfully synthesized and
Scheme 2 Synthesis of two CB-TE1K1P conjugates: (a) cetuximab-click-PEG4–CB-TE1K1P; (b) cFlFlF–PEG4–CB-TE1K1P.
44 | Chem. Commun., 2014, 50, 43--45
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It should be noted that in the CB-TE1K1P chelator, there is
one chiral center in the pendent arm and two enantiomers (R,R
and S,S) associated with the cross-bridge cyclam, statistically
resulting in four diastereomers. Synthesis of pure enantiomers
is in progress and the stereochemistry effects will be investi-
gated as part of a future study.
In summary, here we reported the design and synthesis of a new
chelator, CB-TE1K1P, containing one methanephosphonate and one
carboxylate pendant group, and its use in preparing bioconjugates
through either traditional SPPS or SPAAC. Rapid and efficient
conjugations with the CB-TE1K1P were demonstrated using
cetuximab, and a neutrophil-targeted peptide, cFlFlF. The resulting
conjugates were rapidly radiolabelled with 64Cu under mild condi-
tions in high SA, and the resulting radiotracers showed improved
serum stability when compared with previously reported 64Cu-
labelled cross-bridged chelates, such as CB-TE1A1P. Therefore, CB-
TE1K1P can serve as an improved BFC for conjugation to small
molecules, peptide and protein-based biomolecules, for labelling
with 64Cu or other copper radionuclides with applications in diag-
nostic imaging, radiotherapy, and/or theranostics.
Fig. 2 (a) 64Cu-labellings of three cetuximab–chelate conjugates at
40 1C; (b) serum stability of two cetuximab–chelate conjugates.
Fig. 3 DFT optimized structure of the hypothetical model complex of
64Cu–CB-TE1K1P and 64Cu–CB-TE1A1P.
Supported by NCI R01CA093375.
Notes and references
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The CB-TE1K1P–peptide was radiolabelled under much milder con-
ditions in a shorter time (Fig. S2a, ESI†) compared to the CB-TE1A1P–
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¨
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Chem. Commun., 2014, 50, 43--45 | 45