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.
Chem. Eur. J. 2011, 17, 10222 – 10225
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10223