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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Chemistry. Author manuscript; available in PMC 2017 March 27.
Published in final edited form as:
Chemistry. 2011 September 5; 17(37): 10222–10225.
Published online 2011 August 4. doi:  10.1002/chem.201101894
PMCID: PMC5367772

Efficient Preparation and Biological Evaluation of a Novel Multivalency Bifunctional Chelator for 64Cu Radiopharmaceuticals

Shuanglong Liu, Ph.D.,a Zibo Li, Ph.D.,a Li-Peng Yap, Ph.D.,a Chiun-Wei Huang, Ph.D.,a Ryan Park, M.S.,a and Peter S. Conti, M.D., Ph.D.*,a

Positron emission tomography (PET) is a powerful imaging technique that provides in vivo information on the distribution 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 stratification, 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 evaluating bioactive ligands with long in vivo circulation time. 64Cu (t1/2 = 12.7 h) decays by β+ (20%) and β emission (37%), as well as electron capture (43%), making it well suited for radiolabeling proteins, antibodies and peptides, both for PET imaging (β+) and therapy (β+ and β).[2] The low β+-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]

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 bifunctional chelators (BFCs) for 64Cu. Currently, 1,4,7,10-tetra-azacyclododecane-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-targeted organ radiation dosage and lower the tumor-to-nontumor contrast.[4, 5] 64Cu-Labeled radiopharma-ceuticals with improved stability have been reported including 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivatives,[67] cross-bridged 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (CB-TETA),[5, 8] and 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) derivatives.[911] For these BFCs, relatively harsh conditions such as elevated temperature were generally required for 64Cu chelation. Recently, a 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 demonstrated great in vivo stability and efficient radiolabeling efficiency under mild conditions.[1216] By modifying one of the inert primary amines of sarcophagine, a carboxyl functionalized Sar (AmBaSar, Scheme 1) has been successfully developed in our laboratory.[1214] As sarcophagine has two relatively inert primary amines 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 demonstrated that the multimer of a bioactivate 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 conjugated to multiple targeting ligands via biologically stable amide bonds. In order to prove the advantage of the multifunctional Sar chelators, we chose c(RGDyK) peptide (denoted as RGD), a well-known ligand targeting integrin αvβ3, for the construction of a divalent PET imaging probe.

Scheme 1
The structure of AmBaSar and the synthetic scheme for BaBaSar. Chloride anions were omitted for clarity.

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 (condensation, reduction, demetalation, and deprotection), which also included cation exchange purification and other complicated purification procedures.[1214] The accumulated yield for AmBaSar was approximatly 10% from compound 2.[12] In our initial approach, we tried to obtain BaBaSar by simply increasing the stoichiometry of methyl 4-formylbenzoate in order to introduce another benzoic acid moiety to AmBaSar. However, the synthesis became very difficult due to the multistep reactions and complicated crude compounds obtained. After testing different approaches, we found direct alkylation (SN2) would be an efficient method for the synthesis of BaBaSar. As shown in Scheme 1, the synthesis of compound 2 followed the protocol developed in our laboratory,[1214] which could then be directly alkylated with 4-bromomethylbenzoic acid to afford the product 4,4'-((3,6,10,13,16,19-hexaazabicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene))dibenzoic acid (BaBaSar) in 36% yield. The monoalkylation product (AmBaSar) was also isolated in 30% yield.

After we obtained the bi-functionalized BaBaSar, its free carboxylic acids were activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysulfosu-ccinimide (SNHS) and then conjugated to c(RGDyK) in the presence of N,N-diisopropylethylamine (DIPEA). After HPLC purification, BaBaSar-RGD2 was obtained in 78% yield (Scheme 2). The BaBaSar-RGD2 was labeled with 64Cu very efficiently in 0.1 M NH4OAc buffer within 5 min at room temperature. The radiochemical yield (RCY) was as high as 90.7 ± 5.1% (n = 4). The specific activity of 64Cu-BaBaSar-RGD2 was estimated 200–500 mCi/µmol (5.4–13.5 GBq/µmol). The in vitro stability of 64Cu-BaBaSar-RGD2 was evaluated after 1 h, 4 h, and 20 h incubation in 1×PBS by radioHPLC (Figure S4 in the Supporting Information). Free 64Cu was not detected by radioHPLC up to 20 h. These data are consistent with the previously published stability results.[1216] 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 the blood, tumor, liver, and kidneys were only 38%, 87%, 34%, and 74% for 64Cu-DOTA-RGD at 1 h post injection, respectively.[13] These results further demonstrated the advantages of BaBaSar over DOTA in constructing 64Cu-radiopharma-ceuticals.

Scheme 2
The chemical structures of RGD. The corresponding cartoon structures of 64Cu-BaBaSar-RGD2 and 64Cu-AmBaSar-RGD2.

The competitive U87MG cell-binding assay (IC50) was used to determine the receptor αvβ3 binding affinity of BaBaSar-RGD2, in which 125I-echistatin was employed as a αvβ3-specific radioligand (Figure S6 in the Supporting Information). 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 6.0 ± 0.9 nM and 8.6 ± 1.2 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.

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 via tail vain into 6–7 weeks old nude mice bearing U87MG tumors on the right shoulder. U87MG tumors were clearly visualized at all the time points examined (Figure 1). Region-of-interest (ROI) analysis on microPET images shows the tumor uptakes are 6.16 ± 0.88, 6.22 ± 1.42, and 5.54 ± 1.27 %ID/g at 1, 4, and 20 h post injection, respectively (Figure 2A). The tumor/liver, tumor/kidneys, and tumor/muscle ratios reached 2.99 ± 0.46, 3.03 ± 1.19, and 20.27 ± 6.16 at 20 h post injection, respectively. As a consequence, the high tumor to nontumor ratio provided good contrast for PET imaging.

Figure 1
Decay-corrected whole-body microPET images of athymic female nude mice bearing U87MG tumor from a static scan at 1 h, 4 h, and 20 h after injection of 64Cu-BaBaSar-RGD2, with or without c(RGDyK) as blocking agent (10 mg/kg body weight). Tumors are indicated ...
Figure 2
MicroPET quantification of tumors and major organs at 1 h, 2 h, and 20 h after injection of 64Cu-BaBaSar-RGD2. A. Without blocking agent. B. Coinjection with RGD as blocking agent (10mg/kg body weight).

It is interesting to point out that 64Cu-AmBaSar-RGD2 (the two RGDs were introduced to the same side of the Sar cage, Scheme 2) gave significantly lower tumor uptakes values (P < 0.05) which were 3.04 ± 0.25, 3.15 ± 0.21, and 2.45 ± 0.15 %ID/g at 1, 4, and 20 h post injection, respectively.[18] The dramatic difference of these two otherwise similar structures might be due to the distance between the two RGD motifs. In the BaBaSar-RGD2, there are 22 covalent bonds between two RGDs while there are only 5 covalent bonds between the two RGDs in the AmBaSar-RGD2. The distance between the two cyclic RGD motifs in AmBaSar-RGD2 is probably too short for simultaneously binding to two αvβ3 integrins (Scheme 2). Since the two RGD ligands in BaBaSar-RGD2 have much longer distance and more flexibility, it may be more able to interact with two integrin receptors in cell surface simultaneously. The much higher tumor targeting efficiency of 64Cu-BaBaSar-RGD2 also led to significantly higher tumor to nontumor ratios than those of 64Cu-AmBaSar-RGD2. For example, The tumor/liver was only 0.86 ± 0.10 at 20 h post injection for 64Cu-AmBaSar-RGD2, compared with 2.99 ± 0.46 for 64Cu-BaBaSar-RGD2. This difference further demonstrated the superior properties of BaBaSar in constructing 64Cu radiopharmaceuticals.

Blocking experiments were performed to confirm the integrin αvβ3 specificity of 64Cu-BaBaSar-RGD2. In the presence of a blocking dose of c(RGDyK), the U87MG tumor uptake was reduced to the background level and the uptake values were 0.69 ± 0.12, 0.29 ± 0.05, and 0.13 ± 0.05 %ID/g 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 c(RGDyK) (Figure 2B).

In conclusion, we have successfully demonstrated that the Sar cage could be efficiently functionalized through an alkylation reaction. The cage-like BaBaSar structure demonstrated 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 analog reflects the advantages of the BaBaSar scaffold. In this paper, c(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.

Supplementary Material

Supporting Information


This work was supported by the USC Department of Radiology, the Department of Energy (DE-SC0002353), the National Cancer Institute (P30CA014089), and the USC Biomedical Imaging Science Initiative.


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