New Macrobicyclic Chelator for the Development of Ultrastable 64Cu

Feb 14, 2012 - Luís M. P. Lima , Zakaria Halime , Ronan Marion , Nathalie Camus , Rita Delgado ... Melissa Roger , Luís M. P. Lima , Mathieu Frindel...
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New Macrobicyclic Chelator for the Development of Ultrastable 64 Cu-Radiolabeled Bioconjugate Darpan N. Pandya,† Ajit V. Dale,† Jung Young Kim,‡ Hochun Lee,§ Yeong Su Ha,† Gwang Il An,‡ and Jeongsoo Yoo*,† †

Department of Molecular Medicine, 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 ‡

S Supporting Information *

ABSTRACT: Ethylene cross-bridged cyclam with two acetate pendant arms, ECB-TE2A, is known to form the most kinetically stable 64Cu complexes. However, its usefulness as a bifunctional chelator is limited because of its harsh radiolabeling conditions. Herein, we report new cross-bridged cyclam chelator for the development of ultrastable 64Cu-radiolabeled bioconjugates. Propylene cross-bridged TE2A (PCB-TE2A) was successfully synthesized in an efficient way. The Cu(II) complex of PCB-TE2A exhibited much higher kinetic stability than ECB-TE2A in acid decomplexation studies, and also showed high resistance to reduction-mediated demetalation. Furthermore, the quantitative radiolabeling of PCB-TE2A with 64Cu was achieved under milder conditions compared to ECB-TE2A. Biodistribution studies strongly indicate that the 64Cu complexes of PCB-TE2A cleared out rapidly from the body with minimum decomplexation.

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stability of 64Cu labeled ECB-TE2A complexes, the 64Cu radiotracers derived from ECB-TE2A showed much lower background noise and faster body clearance compared to their counterparts using other BFCs.30−34 However, its usefulness as a BFC is limited because of two major drawbacks. First, its synthesis is not easy: a total of 6 steps involving a total reaction time of 35 days leading to 45% overall yield from cyclam.22 Another big obstacle is its harsh 64Cu labeling conditions. Even though its 64Cu complexes show very high stability after radiolabeling, ECB-TE2A can be radiolabeled with 64Cu only at elevated temperatures of at least 75 °C, at which most heatsensitive biomolecules are degraded.20,35 In an attempt to develop new BFCs, we designed a new class of cross-bridged macrocyclics using ECB-TE2A as a starting point. Even though several ECB-TE2A derivatives having different macrocyclic backbones and N-substituents were reported previously,23−29 no cyclam derivative based on modification of the ethylene-bridge has been previously reported. Here, we increased the length of the cross-bridge from ethylene to propylene in the hope of achieving easier fit of the Cu(II) ion into the chelator, but tighter retention after complexation. Propylene cross-bridged TE2A (PCB-TE2A) was successfully synthesized in five steps starting from cyclam using a transalkylation/cross-bridge reaction/deprotection strategy. The regioselective trans-disubstitution of the tert-butyl acetate groups on the nonadjacent N-atoms of cyclam was achieved

ifunctional chelators (BFCs), which form stable complexes with metal ions, are an essential component for the successful development of metal-based imaging and therapeutic agents.1,2 This is especially the case in the field of nuclear imaging and radioimmunotherapy, where significant advances have been made during the past few decades.3,4 Among the various available radiometal ions, radioactive copper ions (60Cu, 61Cu, 62Cu, 64Cu, 67 Cu) have received a great deal of interest because of their attractive decay characteristics and half-life for medical imaging as well as therapy.5,6 Many 64Cu-radiolabeled bioconjugates have been developed employing several existing BFCs, and some of them are currently in the clinical trial stage.7−9 So far, tetraazamacrocycles, such as DOTA10−13 and TETA,14−17 have commonly been chosen as a BFC for chelating 64Cu ions, but several previous studies demonstrated that 64Cu-DOTA and 64Cu-TETA complexes are not stable under physiological conditions, but they release some free copper ions within several hours.18−21 These released 64Cu ions increase the background noise in positron emission tomography (PET) imaging and also increase the amount of unnecessary radiation exposure in nontargeted organs. In order to address this stability issue, many acyclic and macrocyclic BFCs have been synthesized and tested, but ethylene cross-bridged cyclam with two acetate pendant arms, ECB-TE2A, was found to form the most kinetically stable Cu(II) complexes.7−9 ECB-TE2A was first introduced in 2000 by Weisman et al.22 and various derivatives have since been synthesized,23−29 but it still produces the most kinetically stable Cu(II) complexes known.23,25,26 In particular, its cyclen counterpart, ECB-DO2A, exhibited much lower kinetic stability than ECB-TE2A when complexed with 64Cu.20 Thanks to the high in vivo kinetic © 2012 American Chemical Society

Received: October 1, 2011 Revised: February 8, 2012 Published: February 14, 2012 330

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in quantitative yield via the bisaminal protection of four N-atoms followed by the alkylation of 4 equiv tert-butyl bromoacetate groups.21 Due to its ionic character, compound 3 was insoluble in CH3CN and, therefore, was isolated quantitatively by simple filtration. There was no indication of the formation of cisdisubstituted byproducts in 13C NMR spectrum during the course of the reaction.36 The selective cleavage of the bisaminal bonds of compound 3 was carried out simply by basic hydrolysis using 3 M NaOH to afford the trans-disubstituted cyclam 4 in quantitative yield. The key step, the propylene cross-bridging reaction, had to be optimized. To the best of our knowledge, only one propylene cross-bridged tetraazamacrocylic compound, PCB-cyclen, has been reported by Springborg in 1995, and recently, Wong et al. reported another derivative PCBDO2A.37,38 The Cu complex of PCB-DO2A shows higher in vitro stability than Cu-ECB-TE2A. However, its usefulness as a potential chelator for Cu ions is limited because of its harsh radiolabeling conditions and poor in vivo stability. A mixture of the disubstituted cyclam 4 and 1,3-propanediol di-p-tosylate was refluxed in dried toluene in the presence of K2CO3. By changing the solvent from acetonitrile, in a previous report, to toluene and increasing the reaction temperature, the reaction time was reduced dramatically from six to two days. The tosylate counteranion was removed by treatment with 20% NaOH, and the crude product was column purified to yield the salt-free form of compound 5 in 70% yield. The deprotection of the tert-butyl ester groups of the intermediate 5 was carried out by acidic hydrolysis using 6 M HCl to give the final product, PCB-TE2A, in the form of the hydrochloric salt. By designing an efficient

synthetic route, all of the intermediates and products except for compound 5 were isolated in quantitative yield without tedious column purification. The total reaction time was less than 5 days and the overall yield from cyclam reached 62%, which is superior compared to the reaction time of 35 days and overall yield of 45% in the synthesis of ECB-TE2A. All of the spectroscopic data of PCB-TE2A exactly matched the expected values (see the Supporting Information). The reflux reaction of PCB-TE2A with Cu(ClO4)2·6H2O in the presence of NaOH in methanol afforded the expected Cu-PCB-TE2A complexes in 81% yield. The Cu(II) complex of ECB-TE2A was also prepared according to the previously reported procedure22 for use in direct comparison studies. Then, in order to evaluate the kinetic stability of the two Cu(II) complexes, which is known to be a more critical factor for the stability of Cu complexes under physiological conditions than their thermodynamic stability,8,39 acidic decomplexation and cyclic voltammetry experiments were carried out.25,40 Acidic decomplexation studies, although conducted at conditions markedly different from physiological conditions, are known to provide a good indication of in vivo stability of metal complexes.7,25 The complex, Cu-PCB-TE2A showed exceptionally high stability under highly acidic conditions. Even though the half-life of the Cu-TETA complex in 5 M HCl at 90 °C is only 4.7 min,21 the Cu-PCB-TE2A sample did not show any absorption change in a UV spectrophotometer under the same conditions. Therefore, we carried out the same studies under more harsh conditions, viz., in 12 M HCl at 90 °C, and the Scheme 3. Radiolabeling of PCB-TE2A with 64Cu

Scheme 1. Some Commonly Used Bifunctional Chelators

Scheme 2. Synthesis of Propylene Cross-Bridged TE2A (PCB-TE2A)

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decided to use more reliable 0.2 M phosphate buffer which maintains pH 7 throughout the whole measurement. The extremely high in vitro stability of Cu-PCB-TE2A encouraged us to further examine the 64Cu-radiolabeling and in vivo stability of the complexes. Although the Cu-PCB-TE2A complexes were prepared using the strong base NaOH at reflux condition in good yield, the radiolabeling of PCB-TE2A with 64 Cu should be carried out under milder conditions because the targeting biomolecules conjugated to this BFC could be degraded under these harsh complexation conditions.6 First, we tested the reported sophisticated but time-consuming labeling methods used for the 64Cu radiolabeling of ECBTE2A: 30 min preincubation of chelator using Cs2CO3 in EtOH at 75 °C followed by centrifugation, and then another 30 min incubation with 64Cu at 75 °C.20 Both the PCB-TE2A and ECB-TE2A chelators were radiolabeled with 64Cu in quantitative yield (Figure 2). Then, we tried simple 0.1 M sodium acetate buffer adjusted to pH 8 as a labeling medium. Unexpectedly, PCB-TE2A was radiolabeled with 64Cu in 89% yield at 70 °C within 10 min, and the labeling yield reached 100% after 1 h (Figure 3). However, when ECB-TE2A was used, the same labeling conditions yielded at least two radiolabeled peaks in addition to the free copper peak in radio-TLC (silica, MeOH/10% NH4OAc = 1/1), but actually several byproduct peaks in the radioHPLC analysis (Xbridge C18, 4.6 × 150 mm). The labeling yield of Cu-ECB-TE2A was measured to be only 45% after 1 h of incubation under the same conditions (Figure 3). In the following radiolabeling studies at lower temperatures, PCB-TE2A consistently showed better 64Cu-labeling capacity in the sodium acetate buffer than ECB-TE2A. One hour incubation of PCB-TE2A with 64Cu at 50 °C yielded 64Cu-PCB-TE2A in 89% yield, and the labeling yield reached 58% after 90 min of incubation, even at 40 °C. In contrast, the labeling yield was only 25% at 40 °C after 90 min incubation of ECB-TE2A with 64Cu (Figure 3). As expected from its extremely high in vitro kinetic stability, 64 Cu-PCB-TE2A did not show any sign of decomplexation for up to 24 h in the serum stability test performed at 37 °C. The in vivo behavior and body clearance pattern of 64Cu-PCBTE2A was compared with those of 64Cu-ECB-TE2A in the biodistribution studies in Sprague−Dawley rats (Figure 4). Both complexes showed rapid clearance from the body.

degradation pattern was monitored using an HPLC system (Figure 1a,b). Surprisingly, Cu-PCB-TE2A did not show any sign of degradation for up to seven days (Figure 1a). In contrast, more than 75% of Cu-ECB-TE2A was decomposed after 3 h and less than 3% of intact Cu-ECB-TE2A complex remained at 8 h post-heating in 12 M HCl (Figure 1b). The faster decomposition rate of Cu-ECB-TE2A could also be simply monitored by the color change of its solution from light blue to greenish yellow after 1 h of incubation under the same conditions, while the light blue color of the Cu-PCB-TE2A solution remained exactly the same even after seven days (see the Supporting Information, Figure S2). One important decomplexation mechanism of Cu(II) macrocyclic complexes is the reduction of Cu(II) ions to Cu(I) followed by the demetalation of the free copper ions from the chelators.8,25,40 By measuring the reduction/oxidation behavior of the Cu(II) complexes using cyclic voltammetry, their resistance to in vivo demetalation by reduction could be anticipated. In our experiments, the two complexes, Cu-PCB-TE2A and Cu-ECB-TE2A, showed very different cyclic voltammograms in 0.2 M phosphate buffer at pH 7 (Figure 1c,d). Cu-PCB-TE2A yielded quasi-reversible reduction/oxidation peaks (E1/2 = −0.80 V, ΔE = 80 mV) in addition to a negligible oxidation peak of free Cu(I) ions to Cu(II) at −0.06 V (vs Ag/AgCl), which implies that the Cu-PCB-TE2A holds Cu(I) ion in the coordination geometry and the demetalation is quite suppressed upon the reduction of Cu(II)-PCB-TE2A to Cu(I)-PCB-TE2A. In contrast, Cu-ECB-TE2A showed an irreversible reduction peak at −1.07 V without any oxidation peak responsible for reoxidation of intact Cu(I)-ECB-TE2A. Instead, the significant oxidation current was observed at −0.04 V, which is obviously due to the oxidation of free Cu(I) to Cu(II). These direct comparison studies clearly reveal that Cu-PCB-TE2A is much more resistant to the demetalation upon the reduction than Cu-ECB-TE2A, although Cu-PCB-TE2A is more subject to the reduction by a factor of 230 mV than Cu-ECB-TE2A. Previously, Cu-ECB-TE2A was also reported to show quasi-reversible behavior in 0.1 M sodium acetate solution adjusted to pH 7.40 However, we observed significant pH rise in this buffer solution after the voltammetric measurement, and

Figure 1. Time-dependent UV-HPLC chromatograms of Cu-PCB-TE2A (a) and Cu-ECB-TE2A (b) during acidic decomplexation in 12 M HCl at 90 °C. Cyclic voltammograms (scan rate 100 mV/s, 0.2 M phosphate buffer, pH 7) of Cu-PCB-TE2A (c) and Cu-ECB-TE2A (d). 332

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Figure 4. Biodistribution data of 64Cu-radiolabeled PCB-TE2A and ECB-TE2A at 30 min, 4 h, and 24 h (n = 5).

double liver uptake than 64Cu-ECB-TE2A (3.20 ± 0.28 vs 1.56 ± 0.32%ID/organ) at 30 min postinjection but cleared out gradually from the liver (1.22 ± 0.20%ID/organ at 4 h), a comparable uptake pattern was observed in the kidneys (1.86 ± 0.36 vs 1.57 ± 0.13%ID/organ at 30 min), which could be attributed to the greater lipophilicity of the propylene crossbridge compared to the ethylene cross-bridge. The partition coefficient experiment (octanol/water) confirmed greater lipophilicity of 64Cu-PCB-TE2A to 64Cu-ECB-TE2A. The log P value of 64Cu-PCB-TE2A was measured to be higher than that of 64Cu-ECB-TE2A (−2.84 ± 0.03 vs −3.08 ± 0.04).

Figure 2. UV-HPLC chromatogram of nonradioactive Cu-PCB-TE2A complexes (top) and radio-HPLC chromatogram of 64Cu-PCB-TE2A (bottom).

The percent injected dose per organ (%ID/organ) in all of the collected organs was lower than 1%, except in the liver, at 4 h postinjection. Whereas 64Cu-PCB-TE2A showed more than

Figure 3. UV-HPLC chromatogram (280 nm) of nonradioactive Cu-PCB-TE2A (a) and Cu-ECB-TE2A complex (b); radio-HPLC chromatogram of 64 Cu-PCB-TE2A (c) and 64Cu-ECB-TE2A (d) at 70 °C (1 h incubation) and radio-HPLC chromatogram of 64Cu-PCB-TE2A (e) and 64Cu-ECB-TE2A (f) at 40 °C (90 min incubation). 333

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Prof. Hochun Lee. The Korea Basic Science Institute (Daegu) is acknowledged for the NMR and HR-Mass measurements.

Except for liver, the Cu(II) complexes of PCB-TE2A and ECB-TE2A showed very similar %ID/organ values in all of the organs. At the 24 h time point, the counted activities in all of the organs, except for the liver and kidneys, were at the background level. The liver uptake of 64Cu-PCB-TE2A was decreased dramatically to only 0.12 ± 0.01%ID/organ, which is even lower than the value of 0.18 ± 0.04%ID/organ for 64Cu-ECB-TE2A. The kidneys also showed lower remaining activity in the case of 64 Cu-PCB-TE2A compared to 64Cu-ECB-TE2A (0.038 ± 0.005 vs 0.055 ± 0.005%ID/organ). These biodistribution data imply that 64Cu-ECB-TE2A cleared out at least as fast as 64Cu-PCBTE2A with minimum transchelation of free copper ions from the chelators to biomolecules.18,41 The PCB-TE2A can be used as a bifunctional chelator by its direct conjugation with peptide or antibody just as ECB-TE2A was conjugated with various biomolecules.26,30,42 Even though the direct conjugation of PCB-TE2A with biomolecules using one acetate pendent arm would work well, more versatile conjugation strategy could be achieved by straightforward pendant arm modification.43 In conclusion, we describe a straightforward means of synthesizing propylene cross-bridged TE2A. The Cu(II) complex of PCB-TE2A exhibited extremely robust stability in the acidic decomplexation studies and also showed high resistance to demetalation in the cyclic voltammograms. In contrast to the ethylene cross-bridged TE2A, PCB-TE2A was radiolabeled with 64 Cu in simple sodium acetate buffer in good yield even at low temperatures. Our biodistribution data strongly implies that the decomplexation of 64Cu-PCB-TE2A is minimized under physiological conditions and that the intact 64Cu-PCB-TE2A is cleared from the body at least as fast as 64Cu-ECB-TE2A. The above results demonstrate that PCB-TE2A has greater potential as a BFC than ECB-TE2A. We hope that many radioactive copper-based diagnostic and therapeutic bioconjugates would be prepared employing PCB-TE2A as a BFC and utilized in biomedical studies as well as clinics. Ultrahigh stability nature of Cu-PCB-TE2A would encourage the synthesis of various metal complexes utilizing the related chelators, which could find further application in catalyst and material sciences.

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ASSOCIATED CONTENT

S Supporting Information *

Syntheses and spectroscopic data of compounds 4, 5, and 6, acid decomplexation studies, electrochemical studies, radiolabeling studies of PCB-TE2A and ECB-TE2A, table of biodistribution data. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Fax: +82-53-426-4944. Tel: +82-53-420-4947. E-mail: yooj@ knu.ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Nuclear R&D (grant code: 20090081817, 20110030161) and BAERI (grant code: 20090078235) Programs of NRF, funded by MEST, and the Brain Korea 21 Project in 2011. The CV studies were supported partially by MEST & DGIST (11-BD-0405) for 334

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