Letter pubs.acs.org/acsmedchemlett
Phosphonate Pendant Armed Propylene Cross-Bridged Cyclam: Synthesis and Evaluation as a Chelator for Cu-64 Nikunj Bhatt,† Nisarg Soni,† Yeong Su Ha,† Woonghee Lee,† Darpan N. Pandya,† Swarbhanu Sarkar,† Jung Young Kim,‡ Hochun Lee,§ Sun Hee Kim, ∥ Gwang Il An,‡ and Jeongsoo Yoo*,† †
Department of Molecular Medicine, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University, Daegu 700-422, South Korea ‡ Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, South Korea § Department of Energy Systems Engineering, Daegu Gyeongbuk Institute of Science & Technology, Daegu 711-873, South Korea ∥ Western Seoul Center, Korea Basic Science Institute, Seoul 120-140, Korea S Supporting Information *
ABSTRACT: A propylene cross-bridged macrocyclic chelator with two phosphonate pendant arms (PCB-TE2P) was synthesized from cyclam. Various properties of the synthesized chelator, including Cu-complexation, Cu-complex stability, 64 Cu-radiolabeling, and in vivo behavior, were studied and compared with those of a previously reported propylene crossbridged chelator (PCB-TE2A). KEYWORDS: Bifunctional chelator, propylene cross bridge, phosphonate pendant arms, radiopharmaceuticals
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the acid decomplexation assay. Moreover, in comparison with CB-TE2A, PCB-TE2A has the advantages of milder radiolabeling conditions and easy modification for bioconjugation (Figure 1).
he development of target-specific radiopharmaceuticals using Cu(II) ions requires the use of bifunctional chelators (BFCs). A wide range of acyclic and macrocyclic chelators have been utilized to coordinate Cu(II) ion.1−8 Our group has introduced various types of azamacrocyclic chelators used to form stable Cu(II) complexes, including TE2A,9 TE2A-Bn-NCS,10 MM/DM-TE2A,11 PCB-TE2A,12 and PCB-TE2A-NCS.13 Among these azamacrocyclic chelators, PCB-TE2A was found to form the more stable Cu-complex, which was better than that formed by the chelator CB-TE2A. However, rapid radiolabeling of BFCs is recommended to prevent or minimize radiolytic damage of targeting vector in conjugates,14 and the harsh radiolabeling conditions and slow labeling kinetics of the azamacrocyclic chelators restrict the use of such BFCs in diagnostic and therapeutic applications. The ability of BFCs to coordinate Cu(II) ions is improved by replacing the acetic acid pendant arm with a phosphonate pendant arm, resulting in copper complexes with improved physical properties.15−19 To take advantage of fast metal binding kinetics, cross-bridged BFCs having a phosphonate pendant arm (CB-TE2P and CB-TE1A1P) have been introduced.20,21 Chelators CB-TE2P and CB-TE1A1P were radiolabeled with 64 Cu at room temperature. Unfortunately, the advantages of the changed pendant arm were overshadowed by decreased Cucomplex stability. Although CB-TE2P and CB-TE1A1P form Cu(II) complexes within 5 min at ambient temperature, the stability of the Cu-CB-TE2P and Cu-CB-TE1A1P complexes decreased dramatically.20−22 In comparison with Cu complexes formed using CB-TE2A, Cu-PCB-TE2A complexes had better Cu-complex stability in © XXXX American Chemical Society
Figure 1. Various cross-bridged Cu-chelators described in this letter.
Modification of PCB-TE2A into PCB-TE2P by replacing its acetic acid pendant arms with phosphonate would be expected to retain higher Cu-complex stability in comparison with those formed using CB-TE2A. The propylene cross-bridge of PCBTE2P confers high stability on Cu-complexes, whereas the phosphonate pendant arm may allow radiolabeling under milder conditions and with faster kinetics in comparison with those associated with PCB-TE2A radiolabeling, as was observed in the case of CB-TE2P/TE1A1P. To study our hypothesis, we synthesized a chelator with a propylene cross-bridge and phosphonate pendant arm, which was designated as PCB-TE2P. Overall, PCB-TE2P was synthesized from cyclam in six steps. To prepare PCB-TE2P (2) from PCB-cyclam (1), a single-step Received: September 11, 2015 Accepted: October 14, 2015
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DOI: 10.1021/acsmedchemlett.5b00362 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
ACS Medicinal Chemistry Letters
Letter
spin of 63,65Cu(I = 3/2), are well resolved with the hyperfine value of A∥ = 166 G. The continuous wave electron paramagnetic resonance (CW-EPR) of Cu-PCB-TE2P indicates that the Cu-PCB-TE2P has one paramagnetic copper species of octahedral geometry with nitrogen and oxygen atoms coordinated with the copper ion.5,26 This EPR spectrum complies with the expected structure of Cu-PCB-TE2P. Although acid decomplexation studies of Cu-complexes are not useful for predicting in vivo stability, this type of study provides useful information on the relative strength of the Cucomplex, as it can be performed in various strengths of acid and at various temperatures.9,12 First, the synthesized Cu-complex was evaluated in the acidcatalyzed half-life determination assay by using a spectrophotometer. After acid decomplexation of Cu-PCB-TE2P in 5 M HCl at 90 °C, it did not show any change in absorption, even after 48 h, which strongly indicated that Cu-PCB-TE2P was highly stable under these conditions and suggested that harsher conditions were required for Cu-decomplexation. In comparison with the propylene cross-bridged chelators, ethylene crossbridged chelators showed rapid degradation under the same conditions (5 M HCl at 90 °C), and the half-lives of CB-TE2P and CB-TE1A1P were 3.8 and 6.8 h, respectively21 (Supporting Information Table S1). To carry out Cu-decomplexation and compare the relative strength of Cu-complexes synthesized with Cu-PCB-TE2P, acid decomplexation was performed in 12 M HCl at 90 °C, and degradation of the Cu-complexes was measured quantitatively by HPLC analysis of the reaction mixture. The conditions used were standard conditions for Cu-decomplexation of propylene cross-bridged chelators.12 The results of the HPLC analysis of the reaction mixture are shown in Figure 3a. Although Cu-
process was used in which phosphonate pendant arms were introduced directly onto the secondary amines (Scheme 1).23 Scheme 1. Synthesis of PCB-TE2P from PCB-cyclam
This single-step process was utilized instead of the typical twostep process in which a phosphonate ester was prepared initially, followed by acid hydrolysis to obtain a phosphonate group.24,25 In the process reported here, PCB-cyclam was directly treated with paraformaldehyde and H3PO3 in 6 M HCl as a solvent. To minimize side product formation (Nmethylation), H 3 PO 3 was used in high excess, while paraformaldehyde was added in small amounts over a period of 2 h. We were unable to completely eliminate side product formation, and purification of the reaction mixture by ion exchange chromatography (strong cation exchange resin (Dowex 50WX8) followed by weakly acidic cation exchange resin (Amberlite CG50, H+ form)) was required to isolate pure PCB-TE2P. The isolation yield of pure chelator was 36%. To check the ability of PCB-TE2P to form a stable Cucomplex, a cold Cu-complex of 2 was prepared. To obtain CuPCB-TE2P, 2 was heated at 90 °C with CuCl2 in water (pH adjusted to 8.0 by addition of 1 M NaOH). Using this method, Cu-PCB-TE2P was synthesized within 30 min with a 90% yield (Scheme 2). At room temperature, the reaction did not proceed Scheme 2. Synthesis of Cu-PCB-TE2P
at all. The recrystallized pure Cu-PCB-TE2P complex was evaluated in EPR study, acidic decomplexation and cyclic voltammetry assays. The EPR spectrum of Cu-PCB-TE2P showed a characteristic axial EPR spectrum with the g∥ = 2.25, g⊥ = 2.05 (Figure 2). The four line hyperfine splittings, arisen from the interaction between the electron spin of Cu2+ (d9, S = 1/2) and the nuclear
Figure 3. Time-dependent UV-HPLC chromatograms of Cu-PCBTE2P in 12 M HCl at 90 °C (a) and cyclic voltammogram of CuPCB-TE2P (scan rate 100 mV/s, 0.2 M phosphate buffer, pH 7) (b).
PCB-TE2P showed degradation under the relatively harsh conditions of the test, the rate of degradation was slow. After 2 d, 73% of the intact Cu-PCB-TE2P was observed in the reaction mixture, and this proportion decreased to
