Efficient preparation and biological evaluation of a novel multivalency bifunctional chelator for 64Cu radiopharmaceuticals

Article abstract of DOI:10.1002/chem.201101894

A novel bifunctional chelator: The sarcophagine motif was used in the development of a novel bifunctional chelator for ⁶⁴Cu radiopharmaceuticals (see figure). The high tumor-targeting efficiency and great stability of the ⁶⁴Cu-BaBaSa

Full text of DOI:10.1002/chem.201101894

DOI: 10.1002/chem.201101894
Efficient Preparation and Biological Evaluation of a Novel Multivalency
64
Bifunctional Chelator for Cu Radiopharmaceuticals
Shuanglong Liu, Zibo Li, Li-Peng Yap, Chiun-Wei Huang, Ryan Park, and
Peter S. Conti*[a]
Positron emission tomography (PET) is a powerful imag-
ing technique that provides in vivo information on the distri-
bution of radiolabeled biomolecules. For example, 2-deoxy-
2-18F-fluoro-d-glucose (18F-FDG) has successfully made PET
a routine clinical practice in cancer diagnose, patient stratifi-
cation, and monitoring the treatment of cancer patients.[1]
The advancement of PET depends on the development of
new radiotracers that will complement 18F-FDG. Although
PET nuclides 11C (t1/2 =20.4 min) and 18F (t1/2 =109.7 min)
have been widely used for the development of PET imaging
probes, their short half-lives set a strong limitation for evalu-
ating bioactive ligands with long in vivo circulation time.
64Cu (t1/2 =12.7 h) decays by b+ (20%) and bÀ emission
(37%), as well as electron capture (43%), making it well
suited for radiolabeling proteins, antibodies and peptides,
both for PET imaging (b+) and therapy (b+ and bÀ).[2] The
low b+ energy also promises a good resolution of down to
1 mm in PET images and guarantees minimal radiation
doses to the patients during imaging scans.[3]
new class of BFCs has been synthesized based on the cage-
like hexaazamacrobicyclic sarcophagine (denoted as “Sar”,
compound 2 in Scheme 1). The resulting 64Cu complexes
Because direct addition of 64Cu into a targeting ligand
(such as peptides and antibodies) is not practical, significant
efforts have been devoted to the development of bifunction-
al chelators (BFCs) for 64Cu. Currently, 1,4,7,10-tetra-azacy-
clododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA) is one of
the most widely used chelators for 64Cu labeling. However,
its moderate in vivo stability would increase the non-target-
ed organ radiation dosage and lower the tumor-to-nontumor
contrast.[4,5] 64Cu-labeled radiopharmaceuticals with im-
proved stability have been reported including 1,4,7-triazacy-
clononane-1,4,7-triacetic acid (NOTA) derivatives,[6–7] cross-
bridged 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic
Scheme 1. Structure of AmBaSar and the synthetic scheme for BaBaSar.
DIPEA=N,N-diisopropylethylamine.
demonstrated great in vivo stability and efficient radiolabel-
ing efficiency under mild conditions.[12–16] By modifying one
of the inert primary amines of sarcophagine, a carboxyl-
functionalized Sar (AmBaSar, Scheme 1) has been success-
fully developed in our laboratory.[12–14] As sarcophagine has
two relatively inert primary amine groups on either end of
its cage, we embarked on a project to develop novel Sar
cage derivatives with multifunctional groups introduced to
both ends. In the last decade, numerous studies have dem-
onstrated that the multimer of a bio-active ligand in one
single scaffold can improve both the cell-specific targeting
efficacy and the tumor-targeting efficiency by several orders
of magnitude.[17] In our first chelator design, we intended to
introduce two pendant carboxyl groups at either end of the
Sar cage (named BaBaSar), which could be further conju-
gated to multiple targeting ligands through biologically
stable amide bonds. In order to prove the advantage of the
acid
ACHTUNGTRENNUNG
(CB-TETA),[5,8]
and
1,4,8,11-tetraazabicyclo-
[6.6.2]hexadecane (CB-TE2A) derivatives.[9–11] For these
BFCs, relatively harsh conditions such as elevated tempera-
ture were generally required for 64Cu chelation. Recently, a
[a] S. Liu,+ Z. Li,+ L.-P. Yap, C.-W. Huang, R. Park, Prof. P. S. Conti
Molecular Imaging Center, Department of Radiology
Keck School of Medicine, University of Southern California
1510 San Pablo Street, Room #350, Los Angeles, CA 90033 (USA)
Fax : (+1)323-442-3253
multifunctional Sar chelators, we chose the cACHTNUTRGNE(UNG RGDyK) pep-
E-mail: pconti@usc.edu
[+] These authors contributed equally to this work.
tide (denoted as RGD), a well-known ligand-targeting integ-
rin avb3, for the construction of a divalent PET imaging
probe.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101894.
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Chem. Eur. J. 2011, 17, 10222 – 10225

COMMUNICATION
We first improved the functionalization approach of the
Sar cage. Previously, the benzoic acid moiety was introduced
to the Sar cage through a four-step procedure (condensa-
tion, reduction, demetalation, and deprotection), which also
included cation exchange purification and other complicated
purification procedures.[12–14] The accumulated yield for Am-
BaSar was approximately 10% from compound 2.[12] In our
initial approach, we tried to obtain BaBaSar by simply in-
creasing the stoichiometry of the methyl 4-formylbenzoate
in order to introduce another benzoic acid moiety to AmBa-
Sar. However, the synthesis became very difficult due to the
multistep reactions and complicated crude compounds were
obtained. After testing different approaches, we found that
direct alkylation (SN2) would be an efficient method for the
synthesis of BaBaSar. As shown in Scheme 1, the protocol
developed in our laboratory was followed for the synthesis
of compound 2,[12–14] which could then be directly alkylated
with 4-bromomethylbenzoic acid to afford the product 4,4’-
are consistent with the previously published stability re-
sults.[12–16] The high stability could be due to the cross-
bridged and cage-like configuration of the Sar structure. We
also studied the metabolic stability of 64Cu–BaBaSar–RGD2
in blood, liver, kidneys, and tumor in nude mice bearing
U87MG glioma xenografts at 1 h post injection. The intact
probe was more than 95% in each examined organ by
HPLC analysis (Figure S5 in the Supporting Information).
On the contrary, the amount of intact tracer in blood,
tumor, liver, and kidneys was only 38, 87, 34, and 74% for
64Cu–DOTA–RGD at 1 h post injection, respectively.[13]
These results further demonstrated the advantages of BaBa-
Sar over DOTA in constructing 64Cu radiopharmaceuticals.
The competitive U87MG cell-binding assay (IC50) was
used to determine the receptor avb3 binding affinity of Ba-
BaSar–RGD2, in which 125I-echistatin was employed as a
avb3-specific radioACHTUNTRGENNUGliACHTUGTNRENNUGgand (Figure S6 in the Supporting Infor-
mation). The IC50 of RGD dimer (RGD2) was measured as
a control. Both BaBaSar–RGD2 and RGD2 inhibited the
binding of 125I-echistatin to U87MG cells in a concentration-
dependent manner. The IC50 values for BaBaSar–RGD2,
and RGD2 were (6.0Æ0.9) and (8.6Æ1.2) nm, respectively
(n=3). As expected, the BaBaSar–RGD2 showed a strong
binding affinity to U87MG cells and the introduction of the
BaBaSar motif had minimal effect on the integrin binding
affinity of the probe.
((3,6,10,13,16,19-hexaazabicycloACTHNUGRTNEUNG[6.6.6]ico-sane-1,8-diylbis(a-
za-nediyl))bis(methylene))dibenzoic acid (BaBaSar) in 36%
yield. The monoalkylation product (AmBaSar) was also iso-
lated in 30% yield.
After we obtained the bi-functionalized BaBaSar, its free
carboxylic acid groups were activated with 1-ethyl-3-(3-di-
methylaminopropyl)-carbodiimide (EDC)/N-hydroxysulfo-
succinimide (SNHS) and then conjugated to cACTHNUTRGENUG(N RGDyK) in
the presence of DIPEA. After HPLC purification, BaBaSar-
RGD2 was obtained in 78% yield (Scheme 2). The BaBa-
Sar-RGD2 was labeled with 64Cu very efficiently in 0.1m
NH4OAc buffer within 5 min at room temperature. The ra-
diochemical yield (RCY) was as high as (90.7Æ5.1)% (n=
4). The specific activity of 64Cu–BaBaSar–RGD2 was esti-
mated to be 200–500 mCimmolÀ1 (5.4–13.5 GBqmmolÀ1). The
in vitro stability of 64Cu–BaBaSar–RGD2 was evaluated
after 1, 4, and 20 h incubation in 1ꢁPBS buffer by radi-
oHPLC (Figure S4 in the Supporting Information). Free
64Cu was not detected by radioHPLC up to 20 h. These data
The in vivo tumor-targeting property of 64Cu–BaBaSar–
RGD2 was evaluated by static microPET scans at 1, 4, and
20 h after injection of 64Cu–BaBaSar–RGD2 through the tail
vain into 6–7 weeks old nude mice bearing U87MG tumors
on the right shoulder. U87MG tumors were clearly visual-
ized at all the time points examined (Figure 1). Region-of-
interest (ROI) analysis on microPET images shows the
tumor uptakes are (6.16Æ0.88), (6.22Æ1.42), and (5.54Æ
1.27)%IDgÀ1 at 1, 4, and 20 h post injection, respectively
(Figure 2A). The tumor/liver, tumor/kidneys, and tumor/
muscle ratios reached (2.99Æ0.46), (3.03Æ1.19), and
(20.27Æ6.16) at 20 h post in-
jection, respectively. As a con-
sequence, the high tumor-to-
nontumor ratio provided good
contrast for PET imaging.
It is interesting to point out
that
64Cu–AmBaSar–RGD2
(the two RGDs were intro-
duced to the same side of the
Sar cage, Scheme 2) gave sig-
nificantly lower tumor uptakes
values (P<0.05) which were
(3.04Æ0.25), (3.15Æ0.21), and
(2.45Æ0.15)% IDgÀ1 at 1, 4,
and 20 h post injection, respec-
tively.[18] The dramatic differ-
ence of these two otherwise
similar structures might be due
to the distance between the
two RGD motifs. In the BaBa-
Scheme 2. Chemical structures of RGD and the corresponding cartoon structures of 64Cu–BaBaSar–RGD2 and
64Cu–AmBaSar–RGD2.
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P. S. Conti et al.
AmBaSar–RGD2. For example, The tumor/liver ratio was
only (0.86Æ0.10) at 20 h post injection for 64Cu–AmBaSar–
RGD2, compared with (2.99Æ0.46) for 64Cu–BaBaSar–
RGD2. This difference further demonstrated the superior
properties of BaBaSar in constructing 64Cu radiopharma-
ceuticals.
Blocking experiments were performed to confirm the in-
tegrin avb3 specificity of 64Cu–BaBaSar–RGD2. In the pres-
ence of a blocking dose of cACTHNUTRGNEUGN(RGDyK), the U87MG tumor
uptake was reduced to the background level and the uptake
values were (0.69Æ0.12), (0.29Æ0.05), and (0.13Æ
0.05)%IDgÀ1 at 1, 4, and 20 h post injection, respectively.
The uptake values in most of the normal organs (e.g., liver,
kidneys, and muscle) were also lower than those without co-
injection of cACTHNUTRGNE(NUG RGDyK) (Figure 2B).
Figure 1. Decay-corrected whole-body microPET images of athymic
female nude mice bearing U87MG tumor from a static scan at 1, 4, and
In conclusion, we have successfully demonstrated that the
Sar cage could be efficiently functionalized through an alky-
lation reaction. The cage-like BaBaSar structure demon-
strated favorable 64Cu-labeling properties and the resulting
64Cu–BaBaSar–RGD2 showed great stability both in vitro
and in vivo. The higher tumor uptake of 64Cu–BaBaSar–
RGD2 compared to its 64Cu–AmBaSar–RGD2 analogue re-
flects the advantages of the BaBaSar scaffold. Herein, c-
20 h after injection of 64Cu–BaBaSar–RGD2, with or without c
ACTHNUTRGNEU(GN RGDyK)
as blocking agent (10 mgkgÀ1 body weight). Tumors are indicated by
arrows.
AHCTUNGTREGUN(NN RGDyK) was employed for proof of principle. In the
future, two different biomarkers could be installed onto the
two pedant arms of BaBaSar for constructing dual targeting
probes. Furthermore, the two reactive sites of BaBaSar
could be used to attach a targeting moiety on one side and
an additional label (for secondary imaging modality) or
therapeutic motif on the other side. We anticipate that this
newly developed method will offer a novel way to construct
multimodality imaging and therapeutic drugs.
Acknowledgements
This work was supported by the USC Department of Radiology, the De-
partment of Energy (DE-SC0002353), the National Cancer Institute
(P30A014089), and the USC Biomedical Imaging Science Initiative.
Figure 2. MicroPET quantification of tumors and major organs at 1, 4,
and 20 h after injection of 64Cu–BaBaSar–RGD2. A) Without blocking
agent. B) Co-injection with RGD as blocking agent (10 mgkgÀ1 body
weight).
Keywords: bifunctional chelators
avb3 · radiopharmaceuticals · RGD · sarcophagine
· copper-64 · integrin
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Preparation and Biological Evaluation of a Chelator for Radiopharmaceuticals
COMMUNICATION
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Received: June 21, 2011
Published online: August 4, 2011
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