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(17, 18) This ECB-TE2A could be used as a BFC, but because the compound does not contain any other functional groups that could be used for conjugatio...
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Propylene Cross-Bridged Macrocyclic Bifunctional Chelator: A New Design for Facile Bioconjugation and Robust 64Cu Complex Stability Darpan N. Pandya,†,∥ Nikunj Bhatt,†,∥ Gwang Il An,‡,∥ Yeong Su Ha,† Nisarg Soni,† Hochun Lee,§ Yong Jin Lee,‡ Jung Young Kim,‡ Woonghee Lee,† Heesu Ahn,† 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 S Supporting Information *

ABSTRACT: The first macrocyclic bifunctional chelator incorporating propylene cross-bridge was efficiently synthesized from cyclam in seven steps. After the introduction of an extra functional group for facile conjugation onto the propylene cross-bridge, the two carboxylic acid pendants could contribute to strong coordination of Cu(II) ions, leading to a robust Cu complex. The cyclic RGD peptide conjugate of PCB-TE2A-NCS was prepared and successfully radiolabeled with 64Cu ion. The radiolabeled peptide conjugate was evaluated in vivo through a biodistribution study and animal PET imaging to demonstrate high tumor uptake with low background.

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INTRODUCTION In recent years, bifunctional chelators (BFCs) have found multiple applications in the field of medical imaging and therapy.1−4 In particular, BFC plays a crucial role in the development of target-specific radiopharmaceuticals in which a targeting biomolecule (e.g., disease-specific peptide or antibody) is linked with a metallic radionuclide by BFC.5−7 The ideal BFC requires the ability to form facile and strong conjugations with biomolecules and to complex with radiometal ions firmly in physiological conditions.8 Among many radiometal ions, 64Cu has becomes increasingly popular due to its attractive decay mode (β+ 17%, β− 39%) and midlong half-life (12.7 h), which allows both positron emission tomography (PET) imaging and radiotherapy.9 In the literature, various BFCs have been used for 64Cu labeling. Most of these compounds are based on DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid), NOTA (1,4,7-triazacyclododecane-N,N′,N″-triacetic acid), or their derivatives. The wide acceptance of polyazamacrocyclic chelators such as BFCs is due to their greater in vivo stability in comparison to acyclic chelators such as EDTA and DTPA.10 However, in recent studies, the in vivo stability of Cu complexes of BFCs have been reported to be unsatisfactory, and the chelating agents lose Cu(II) ions in vivo, which increase the unnecessary radiation exposure to nontargeted organs, as well as background noise.11,12 Therefore, the search for a better BFC still continues. To minimize the decomplexation of Cu-BFCs in vivo and to improve the stability of Cu-BFC complexes, various modifications on the tetraaza-macrocyclic backbone have been © 2014 American Chemical Society

attempted by several research groups. These modifications include the variation of the length of carboxylate pendant arm from the N atoms13 and the addition of extra rigidity by introducing side-bridges14,15 or cross-bridge16 to the cyclic framework. Among these chemical modifications, replacing the transpositioned two acetic acid pendant arms to connect both secondary amines with an ethylene linker and producing a cagelike structure (i.e., ECB-TE2A, Figure 1) was found to be the most effective method to increase the Cu-BFC stability, which ultimately results in a reduction in the trans-metalation of Cu in vivo.17,18 This ECB-TE2A could be used as a BFC, but because the compound does not contain any other functional groups that could be used for conjugation, modification with biomolecules requires the sacrifice of one of its two acetic acid functionalities. The utilization of the acetic acid functionality for conjugation leads to reduced Cu-BFC complex stability, as the group is not available for strong coordination with Cu(II) ions.19 Furthermore, because only one carboxylate group can coordinate to a Cu ion, the overall charge of the complex changes from neutral to +1. Positively charged Cu complexes demonstrate higher uptake in clearance organs such as the liver and kidney, and the body clearance is slow in comparison to that of neutral complexes.14,20 To solve this conjugation problem, ECB-TE2A derivatives in which the tetraaza-macrocyclic backbone or N-substituted pendant was modified by an extra functional group were also introduced. However, the total synthetic steps were drastically increased, Received: March 5, 2014 Published: August 19, 2014 7234

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Figure 1. Structure of the bifunctional chelators.

Scheme 1. Seven-Step Synthesis of PCB-TE2A-NCS from Cyclam

introduced as a form of glyoxal, while the propylene is introduced as a 1,3-propanediol di-p-tosylate form in crossbridge reactions. In this study, we report a highly effective synthetic route for a novel bifunctional chelator incorporating PCB-TE2A, in which an extra functional group (NCS) for facile conjugation is introduced onto the propylene cross-bridge, and the two carboxylate pendants are saved for strong coordination to Cu(II) ions. The chelator, PCB-TE2A-NCS, is conjugated with a cyclic RGD peptide through the NCS functionality of the BFC and radiolabeled with 64Cu ions. The in vivo targeting

and overall yields were significantly decreased, rendering the use of these derivatives impractical.21,22 Recently, we reported that propylene cross-bridged TE2A (PCB-TE2A) could form a more stable Cu complex than ECBTE2A and could be radiolabeled with 64Cu ions in milder conditions.23 In addition, the synthetic process of PCB-TE2A is advantageous, as it can be synthesized in higher overall yield from commercially available cyclam in shorter times, compared with ECB-TE2A. Furthermore, there is a better chance of introducing extra functional groups onto a propylene bridge than an ethylene bridge because the ethylene bridge is 7235

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Scheme 2. Three-Step Synthesis of Intermediate for Propylene Cross-Bridge

affinity of 64Cu-labeled PCB-TE2A-NCS-c(RGDyK) was examined in U87MG tumor models.

This synthetic route of introducing the additional functional group onto the carbon atom of the cross-bridge for facile conjugation is highly advantageous in terms of overall yield and total reaction time in comparison with the previously reported synthetic routes (Figure 1). PCB-TE2A-NCS was synthesized in 60% overall yield starting from the readily available cyclam with total 6 days of reaction time, which is far superior than 7% overall yield with 33 days reaction time for ECB-TE2A-NHS and 9% overall yield with 41 days reaction time for ECB-TE2ANCS (Table 1).21,22 In previously reported syntheses, the extra

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RESULTS AND DISCUSSION PCB-TE2A-NCS was successfully synthesized in seven steps from a commercially available cyclam, as shown in Scheme 1. The trans-dialkylated 1,8-N,N′-bis(carbo-tert-butoxymethyl)1,4,8,11-tetraazacyclotetradecane (4) was synthesized in quantitative yield from cyclam by a regioselective protection/ alkylation/deprotection strategy.23 For the propylene cross-bridge precursor, a suitably tailored nitro-derivative of a ditosyl ester of 1,3-propane diol (11) was synthesized from diethyl malonate in three steps by modifications of reported procedures (Scheme 2).24,25 In brief, a condensation reaction of diethyl malonate with 4-nitro benzyl bromide was carried out in the presence of K2CO3 to afford diethyl 2-(4-nitrobenzyl)malonate (9). In this reaction, a mild base and excess of diethyl malonate were used to prevent the formation of dialkylated byproduct. The intermediate 9 was reduced using sodium borohydride to furnish the diol, which was recrystallized from EtOAc/hexane to generate pure 2-(4nitrobenzyl)propane 1,3-diol (10) as an off-white powder. The alcohol groups of 10 were tosylated by treatment with ptoluenesulfonyl chloride in the presence of pyridine to afford pale yellow oil, which was crystallized from ethanol to yield a white solid (11). The introduction of a propylene cross-bridge with additional functionality to the two newly generated secondary amines was carried out by treating compound 4 with 11 in dried toluene in the presence of anhydrous Na2CO3. The removal of tosylate salts of the obtained brown colored oil was achieved by treatment with 20% NaOH, and the crude product was purified by column chromatography to obtain salt-free compound 5. Deprotection of tert-butyl group was achieved by treating compound 5 with a 1:1 (v/v) mixture of trifluoroacetic acid (TFA) and methylene dichloride at room temperature to afford the TFA salt of PCB-TE2A-NO2 (6) as an oil, which was further triturated with Et2O to provide an off-white solid. For the reduction of the nitro group, 6 was treated with 10% Pd/C in water under H2 (g) at room temperature for 12 h. The resulting reaction mixture was then filtered off and washed with water, and the combined filtrates were evaporated under vacuum to yield an oily residue, which was triturated with Et2O to provide an off-white solid (7). The NH2 group of 7 was substituted by NCS by reaction with thiophosgene in a CHCl3 and 0.5 M HCl solution. The reaction mixture was stirred at room temperature, followed by layer separation and washing of the CHCl3 layer with water. The combined aqueous layers were washed with CHCl3 to remove unreacted thiophosgene. Finally, the aqueous layer was lyophilized to afford white solid (8) (for more information, see the Supporting Information).

Table 1. Comparison of the Syntheses of the Bifunctional Chelators Containing an Extra Functional Group for Facile Conjugation bifunctional chelator

total steps for synthesis

overall yield

total time for synthesis

PCB-TE2A-NCS ECB-TE2A -NHS21 ECB-TE2A -NCS22

7 8

60% 7%

6 days 33 days

13

9%

41 days

functional group was introduced either at the CH2 carbon of the cyclam backbone or by the modifying carboxylic acid pendent arm. The introduction of an extra functional group on the ethylene cross-bridge is difficult because this bridge was introduced by using glyoxal.16 Although we have already proven that PCB-TE2A formed ultrastable Cu complexes as ECB-TE2A, the in vitro stability of the Cu complex of PCB-TE2A-NCS was evaluated to verify the influence of the structural modification on its Cu complex stability. As a model Cu complex, the compound 7 was used instead of PCB-TE2A-NCS (8) because the NCS group of 8 was observed to be converted to NH2 during complexation reaction, which was confirmed by mass analysis of reaction mixture, and the expected Cu-PCB-TE2A-NCS complex could not be isolated. To a solution of PCB-TE2A-NH2 and Cu(ClO4)2·6H2O in methanol, 1 M solution of NaOH was added, and the reaction mixture was refluxed for 2 h, cooled, and filtered through celite bed. The filtrate was subjected to diethyl ether diffusion. The deposited blue crystals were collected and dried to obtain Cu-PCB-TE2A-NH2 in 88% yield. To evaluate the kinetic stability of Cu-PCB-TE2A-NH2, acidic decomplexation and cyclic voltammetry experiments were performed, and the results were compared with those of Cu-PCB-TE2A.13,26 An acidic decomplexation study was carried out in extremely harsh conditions (12 M HCl at 90 °C), and the degradation pattern was monitored by repeated sample injection into an HPLC (Figure 2A,B). As the decomplexation reaction proceeds, the peak area for intact Cu-PCB-TE2A-NH2 at 6.6 min should decrease, and the peak 7236

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Figure 2. Time-dependent UV-HPLC chromatograms of Cu-PCB-TE2A-NH2 (A) and Cu-PCB-TE2A (B) during acidic decomplexation in 12 M HCl at 90 °C. Cyclic voltammograms (scan rate 100 mV/s) of Cu-PCB-TE2A-NH2 (C) and Cu-PCB-TE2A (D).

Scheme 3. Conjugation of PCB-TE2A-NCS to c(RGDyK) Peptide

validated αvβ3 integrin ligand, and its radiolabeling efficiency with 64Cu was also verified. PCB-TE2A-NCS was simply conjugated to c(RGDyK) by treating c(RGDyK) with PCB-TE2A-NCS in 0.1 M Na2CO3 (pH 9.5) and stirring overnight in the dark, followed by purification by semipreparative HPLC (Scheme 3). The collected fraction was combined and lyophilized to afford the final product as white powder in 72% yield. The purified PCBTE2A-NCS-c(RGDyK) was confirmed by analytical HPLC and ESI mass spectrometry [m/z calculated for C52H79N14O12S, 1122.56; found m/z, 1122.49 for [M]−]. The purified PCB-TE2A-NCS-c(RGDyK) conjugate was radiolabeled with 64Cu by incubation with 64CuCl2 in 0.1 M NaOAc buffer (pH 8.0) at 80 °C for 1 h, and the radiochemical yield and purity were confirmed by radio-TLC and HPLC, respectively. The PCB-TE2A-NCS-c(RGDyK) was radiolabeled with 64Cu at >95% radiochemical yield, and the radiochemical purity was over 98% after HPLC purification (Figure 3). The specific activity of 64Cu-PCB-TE2A-NCSc(RGDyK) was calculated to be in the range of 900−1050 μCi/ μg. The logP values calculated for 64Cu-PCB-TE2A-NCSc(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK) were −2.81 and −2.98, respectively, indicating that 64Cu-PCB-TE2A-NCSc(RGDyK) is more lipophilic than 64Cu-ECB-TE2A-c(RGDyK). The purified conjugate was also evaluated for

area for free copper (at 2.0 min) should increase. However, the Cu-PCB-TE2A-NH2 did not demonstrate any decomposition in 12 M HCl even after 7 days, and it remained intact, as observed in the case of Cu-PCB-TE2A. This study indicates that the further alkylation of the propylene cross-bridge did not affect the stability of the Cu complex of the PCB-TE2A core and also strongly implies that this high stability of Cu-PCBTE2A-NH2 complex will be maintained even after conjugation with biomolecules through NCS group, as the conjugated group on the propylene cross-bridge is located far from the Cu coordination core, which contains two carboxylate groups that can coordinate strongly to Cu(II) ions. The electrochemical behavior of Cu-PCB-TE2A-NH2 was also examined by a cyclic voltammetry study to identify any possibility of decomplexation by reducing Cu(II) to Cu(I) in physiological conditions, as the two oxidation states of Cu ions favor different coordination geometries. As shown in Cu-PCBTE2A (Figure 2D), Cu-PCB-TE2A-NH2 generated quasireversible reduction/oxidation peaks (E1/2 = −0.84 V) without any oxidation peak for free Cu(I) ions to Cu(II), which indicates that PCB-TE2A-NH2 can even complex with Cu(I) ions firmly (Figure 2C). After confirming the high in vitro stability of the Cu complex of PCB-TE2A-NH2, we tested the feasibility of conjugating PCB-TE2A-NCS with cyclic RGD peptide, which is a well7237

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of Cu(II) ions from chelator than PCB-TE2A-NCS analogue (24.0% vs 5.5%). In case of liver and kidney samples, though the difference observed is marginal, still 64Cu-PCB-TE2A-NCSc(RGDyK) shows slightly less demetalation of 64Cu ions compared to the 64Cu-ECB-TE2A-c(RGDyK) (10.0% vs 10.6% in liver and 8.7% vs 12.3% in kidney for 64Cu-PCB-TE2A-NCSc(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK), respectively). In addition to free 64Cu ion peaks, only intact 64Cu-PCB-TE2ANCS-c(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK) peaks were seen in HPLC chromatograms (Figure 5). Higher in vivo stability of Cu complex of PCB-TE2A-NCS conjugate could be partially attributed to the availability of both carboxylate pendant arms for the strong coordination to 64Cu. The in vivo behavior and tumor targeting efficacy of 64Culabeled PCB-TE2A-NCS-c(RGDyK) conjugate was evaluated by a biodistribution study in U87MG tumor-bearing nude mice. The biodistribution data for 64Cu-PCB-TE2A-NCS-c(RGDyK) is displayed in Figure 6. First, the PCB-TE2A-NCS-conjugated RGD peptide was rapidly cleared from the body by both the kidney and liver. The blood activity at 1 h after injection was only 0.25 ± 0.03 %ID/g, while the kidney and liver activities were 4.95 ± 0.15 and 3.21 ± 0.28 %ID/g, respectively, at the same time. The tumor also exhibited the second highest uptake after the kidney (3.65 ± 0.08 %ID/g) at 1 h postinjection. The αvβ3 receptor-mediated specific uptake of radiolabeled RGD peptide was confirmed by a blocking study. At the coinjection of cold c(RGDyK) peptide (5 mg/kg), tumor uptake was reduced to one-fourth of that of the nonblocked sample (3.65 ± 0.08 vs 0.90 ± 0.11 %ID/g), while other organs exhibited comparable uptakes. The activities in the clearance organs gradually decreased over time, and about one-third of the 1 h activities were observed in the liver and kidneys at 24 h (1.06 ± 0.21 and 1.86 ± 0.37 %ID/g, respectively). However, the tumor exhibited much more prolonged retention of radiolabeled RGD peptide, mostly because of specific binding of peptide to αvβ3 receptor.29−31 The tumor uptake was even slightly increased at 4 h, and 75% of the activity was remaining at 24 h, compared to 1 h (3.72 ± 0.36 %ID/g at 4 h and 2.73 ± 0.79 %ID/g at 24 h). At 24 h postinjection, the highest activity was found in the tumor (red color in Figure 6). The tumor-to-organ ratios were 46.2, 20.7, 7.2, 4.1, 1.5, and 2.6 for blood, muscle, bone, intestine, kidney, and liver, respectively, at 24 h. Blood and muscle uptake at 24 h was only 0.06 ± 0.01 and 0.14 ± 0.08 % ID/g, respectively. Our 64Cu-PCB-TE2A-NCS-c(RGDyK) demonstrated higher tumor uptakes than previously reported for 64Cu-AmBaSar-c(RGDyK) and 64Cu-DOTA-c(RGDyK)27 but showed comparable tumor uptakes with 64Cu-NODAGAc(RGDfK) and 64Cu-CB-TE2A-c(RGDfK).29 Even though Fani et al. used c(RGDfK) instead of c(RGDyK), it would be worthy to compare their biodistribution data with the current one because they also used the same U87MG tumor model, and ethylene cross-bridged TE2A (CB-TE2A) closely resembles our propylene cross-bridged TE2A (PCB-TE2A). Tumor uptakes of both 64Cu-PCB-TE2A-NCS-c(RGDyK) and 64CuCB-TE2A-c(RGDfK) are very comparable (3.65 ± 0.08 vs 3.66 ± 0.58 at 1 h; 2.73 ± 0.79 (24 h) vs 2.99 ± 0.79 (18h), respectively). Both tracers cleared fast and showed high tumorto-background ratios (tumor-to-blood, 14.6 vs 7.48; tumor-tomuscle, 19.2 vs 13.6 at 1 h, for PCB-TE2A and CB-TE2A analogues, respectively). 64Cu-PCB-TE2A-NCS-c(RGDyK) showed higher liver and kidney uptakes than 64Cu-CB-TE2Ac(RGDfK) at 1 h (liver, 3.21 ± 0.27 vs 1.57 ± 0.54; kidney, 4.95 ± 0.14 vs 2.14 ± 0.53, respectively), while 64Cu-CB-TE2A-

Figure 3. UV-HPLC chromatogram (218 nm) of PCB-TE2A-NCSc(RGDyK) (top) compared with radio-HPLC chromatogram of 64CuPCB-TE2A-NCS-c(RGDyK) (bottom).

serum stability. In fetal bovine serum (FBS), the conjugate did not present any sign of decomposition up to 24 h at 37 °C. To check the effect of conjugation of c(RGDyK) with PCBTE2A-NCS on integrin binding affinity, in vitro affinity assay was carried out by using PCB-TE2A-NCS-c(RGDyK) (Figure 4). The 50% inhibitory concentration (IC50) calculated for

Figure 4. In vitro inhibition of 125I-echistain binding to integrin on U87MG cells by c(RGDyK) and PCB-TE2A-NCS-c(RGDyK).

PCB-TE2A-NCS-c(RGDyK) was comparable with that of reference standard c(RGDyK) (61.5 ± 7.14 and 29.97 ± 5.41 nM, respectively), which indicates that the conjugation of PCBTE2A-NCS with c(RGDyK) has minimal effect on its affinity toward αvβ3 integrin. The in vivo stability study of 64Cu-PCB-TE2A-NCSc(RGDyK) was carried out with little modification in the published procedures.27,28 For the comparison, 64Cu-labeled tracer ECB-TE2A-c(RGDyK) was prepared as per a reported process.29 A comparative HPLC blood, liver, and kidney analysis data of two tracers are displayed in Figure 5. The results clearly showed that 64Cu-PCB-TE2A-NCS-c(RGDyK) is more robust in rat blood and that the demetalation of free 64 Cu ions from PCB-TE2A-NCS chelator is lesser compared to ECB-TE2A analogue. While 64Cu-ECB-TE2A-c(RGDyK) showed 24.0% of demetalated free 64Cu ion peak in rat blood sample at 1 h postinjection, 64Cu-PCB-TE2A-NCS-c(RGDyK) showed only 5.5% of free copper ion peak. 64Cu-labeled ECBTE2A complex showed more than 4 times higher demetalation 7238

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Figure 5. In vivo stability of radiotracers in rat at 1 h postinjection. HPLC profiles of (A) blood, (B) liver, and (C) kidney for 64Cu-PCB-TE2ANCS-c(RGDyK) and (D) blood, (E) liver, and (F) kidney for 64Cu-ECB-TE2A-c(RGDyK). Free 64Cu2+ ions (●), intact 64Cu-PCB-TE2A-NCSc(RGDyK) (▲), and intact 64Cu-ECB-TE2A-c(RGDyK) (■).

Figure 6. Biodistribution data of 64Cu-PCB-TE2A-NCS-c(RGDyK) at 1 h, 1 h blocking, and 4 and 24 h postinjection in U87MG tumor bearing nude mice (n = 3).

c(RGDfK) showed much higher bone uptake than PCB-TE2A counterpart (2.57 ± 0.23 vs 0.33 ± 0.02). Higher liver uptake could be partially attributed to more lipophilicity of benzylNCS linker of PCB-TE2A-NCS chelator32 (logP values: −2.81 and −2.98 for 64Cu-PCB-TE2A-NCS-c(RGDyK) and 64CuECB-TE2A-c(RGDyK), respectively), and higher bone uptake of 64Cu-CB-TE2A-c(RGDfK) could be partially attributed to intrinsic bone uptake property of demetalated free 64Cu ions. All other organ uptakes are comparable. Rapid body clearance and high tumor uptake indicate that PCB-TE2A-NCS chelator complexes well with 64Cu ions and that its conjugation with the RGD peptide through a thiourea bond is stable in physiological conditions. The animal PET imaging data further confirmed the biodistribution data of 64Cu-PCB-TE2A-NCS-c(RGDyK). Coronal PET images collected at 1, 4, and 24 h are shown in Figure 7. The U87MG xenograft tumor on the right flank of nude mouse was clearly observed even at 1 h after injection (3.80 ± 0.25 %ID/g). However, high activities were found in the abdominal region, especially in the liver and intestines (3.74 ± 0.35 and 3.62 ± 0.36 %ID/g, respectively). At 4 h, liver activity was decreased, but more activity was found in the

Figure 7. PET image of 64Cu-PCB-TE2A-NCS-c(RGDyK) at 1, 4, and 24 h postinjection in U87MG tumor-bearing nude mice. White arrow, tumor; yellow arrowhead, liver; green arrowhead, intestine.

intestines (4.67 ± 0.44 %ID/g), which is in good agreement with the biodistribution data. Most of the abdominal activities in the liver, intestine, and kidneys were cleared from body at 24 7239

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h (liver 1.71 ± 0.08, kidney 1.52 ± 0.16, and muscle 0.12 ± 0.01 %ID/g), but the tumor activity was still similar to that observed at 1 h (3.49 ± 0.10 %ID/g at 24h), resulting in unambiguously clear tumor imaging with low background. The advantage of Cu-64 as a radioiostope is its midlong half-life (12.7 h), which allows late imaging at time points when most activities clear from nonspecific organs, leaving only specific binding in the target organ(s) or tissue(s). As observed with our targeted chelator, the tumor can be much more easily identified at later time points than at earlier ones.

(t, 1H, J = 7.7 Hz), 4.13−4.21 (m, 4H), 7.38−7.40 (dd, 2H, J = 10 Hz), 8.13−8.15 (dd, 2H, J = 10 Hz). 13C NMR (125.8 MHz, CDCl3): δ 15.54, 35.85, 54.64, 63.34, 125.27, 131.38, 147.19, 148.50, 169.78. HRMS (FAB) calculated for C14H18NO6, 296.1134 [(M + H)+]; found, 296.1138 [(M + H)+]. Synthesis of 2-[(4-Nitrophenyl)methyl]propan-1,3-diol (10). To a suspension of NaBH4 (14.35 g, 379.3 mmol) in EtOH (200 mL) was added dropwise a solution of 9 (11.2 g, 37.93 mmol) in EtOH (100 mL), and the mixture was refluxed for 16 h. After the addition of aqueous NH4Cl solution in small portions (5%, 150 mL), the crude solution was distilled under vacuum to remove EtOH. CH2Cl2 (200 mL) was added, and the mixture was filtered. When the two layers were separated, the aqueous phase was extracted with CH2Cl2 (3 × 50 mL), and then the combined organic phases were washed with aqueous NaHCO 3 (5%, 100 mL), dried over MgSO4 , and concentrated under vacuum. The resulting yellow oil was recrystallized from EtOAc/hexane to yield the off-white powder of 10 (6.81 g, 85% yield). 1H NMR (500 MHz, CD3OD): δ 1.92−1.98 (m, 1H), 278− 2.79 (d, 2H, J = 6.5 Hz), 3.53−3.54 (d, 4H, J = 5.5 Hz), 7.44−7.46 (dd, 2H, J = 8.5 Hz), 8.12−8.14 (dd, 2H, J = 9 Hz). 13C NMR (125.8 MHz, CD3OD): δ 33.42, 44.93, 61.22, 122.89, 129.80, 146.30, 148.88. HR MS (FAB) calculated for C10H14NO4, 212.0923 [(M + H)+]; found, 212.0923 [(M + H)+]. Synthesis of 2-[(4-Nitrophenyl)methyl]propan-1,3-diol bis(4-methylbenzenesulfonate) (11). A solution of p-toluenesulfonyl chloride (14.51 g, 76.11 mmol) in pyridine (30 mL) was added dropwise to a stirred solution of 10 (6.43 g, 30.44 mmol) in pyridine (20 mL), while the temperature was maintained below 0 °C. After the completion of the addition, the reaction mixture was stirred at room temperature for 10 h, and then it was poured into ice-cold aqueous 5 M HCl solution (100 mL). The aqueous phase was extracted with CH2Cl2 (3 × 100 mL), and then the combined organic phase was washed with brine (2 × 100 mL), dried over MgSO4, and evaporated under reduced pressure to afford pale yellow oil, which was further crystallized from ethanol (100 mL) to obtain the white solid of compound 11 (14.08 g, 89% yield). 1H NMR (500 MHz, CDCl3): δ 2.32−2.34 (m, 1H), 2.46 (s, 6H, 2 × Ar−CH3), 2.72−2.74 (d, 2H, J = 7.5 Hz), 3.85−3.99 (m, 4H), 7.13−7.15 (dd, 2H, J = 8 Hz), 7.33−7.35 (dd, 4H, J = 8.5 Hz), 7.70−7.72 (dd, 4H, J = 8 Hz), 8.00−8.02 (dd, 2H, J = 9 Hz). 13C NMR (125.8 MHz, CDCl3): δ: 21.58, 32.93, 39.66, 67.73, 123.70, 127.82, 129.74 129.97, 132.08, 145.09, 145.34, 146.70. HR MS (FAB) calculated for C24H26NO8S2, 520.1100 [(M + H)+]; found, 520.1097 [(M + H)+]. Synthesis of 4,11-N,N′-Bis(carbo-tert-butoxymethyl)-16-(4nitrobenzyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane (5). A solution of 1,8-N,N′-bis(carbo-tert-butoxymethyl)-1,4,8,11-tetraazacyclotetradecane 4 (5.23 g, 12.20 mmol), 2-[(4-nitrophenyl)methyl]propan-1,3-diol bis(4-methylbenzenesulfonate) 11 (6.34 g, 12.20 mmol) and anhydrous Na2CO3 (2.97 g, 28.06 mmol) in anhydrous toluene (200 mL) was refluxed for 2 days. The solvent was evaporated from the reaction mixture under reduced pressure, and CH2Cl2 (200 mL) was added. The resulting brown slurry was filtered off to remove Na2CO3 through a celite pad and was washed with CH2Cl2 (2 × 25 mL). The solvent was evaporated from the combined filtrate and wash materials under reduced pressure. The residue was dissolved in 20% aq NaOH (100 mL). After stirring for 4 h, the resultant solution was extracted with CHCl3 (3 × 100 mL). The combined extracts were washed by brine and dried over MgSO4, and the solvent was evaporated under reduced pressure. The resulting residue was purified via column chromatography on alumina (basic) and eluted with CH2Cl2/methanol (20:1) to afford the off-white powder of compound 5 (5.45 g, 74% yield). 1H NMR (500 MHz, CDCl3): δ 8.14−8.12 (dd, 2H, J = 9 Hz), 7.60−7.59 (dd, 2H, J = 8.5 Hz), 4.09 (t, 1H, J = 11.5 Hz), 3.64−3.14 (m, 9H), 3.11−2.54 (m, 16H) 2.42−2.20 (m, 3H), 1.74−1.52 (m, 2H), 1.48−1.45 (d, 18H, J = 15 Hz). 13C NMR (125.8 MHz, CDCl3): δ 171.11, 169.84, 147.15, 146.89, 130.44, 123.90, 81.86, 81.68, 61.12, 60.92, 57.59, 56.75, 56.49, 55.25, 52.66, 50.96, 49.54, 47.99, 36.46, 31.44, 28.41, 28.22, 24.13, 22.23. HRMS (FAB) calculated for C32H54N5O6, 604.4074 [(M + H)+]; found, 604.4077 [(M + H)+].

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CONCLUSIONS PCB-TE2A-NCS containing an extra functional group for easy and selective conjugation with biomolecules was synthesized in an efficient way by introducing the NCS group at the propylene cross-bridge. The synthesized PCB-TE2A-NH2 was demonstrated to form robust Cu complexes by keeping both carboxylic acid pendants available for strong complexation with Cu(II) ions. PCB-TE2A-NCS was easily conjugated with peptide, and the prepared conjugate was radiolabeled with 64Cu in high yield. Biodistribution, in vivo stability, and animal-PET imaging studies confirmed the high in vivo stability and excellent tumor targeting efficacy of the RGD peptideconjugated PCB-TE2A-NCS chelator. Various propylene cross-bridged BFCs containing other functional groups for specific conjugation could be developed by employing our synthetic strategy. In addition, the introduction of new linker instead of benzyl group on PCB-TE2A could lead to more favorable physiological properties of 64Cu-labeled tracer.

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EXPERIMENTAL SECTION

Materials and Methods. Cyclam was purchased from CheMatech (Dijon, France). All other reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO) and were used as received. Copper-64 was produced at KIRAMS (Seoul, Korea) by the 64Ni(p,n)64Cu nuclear reaction using an MC50 Cyclotron (Scanditronix, Sweden). Instrumentation. All 1H NMR and 13C NMR spectra were measured on a Varian Unity Inova 500 MHz instrument. Highresolution mass spectra (HRMS) were recorded on a JEOL JMS700 or Quattro Premier XE mass spectrometer. Elemental analyses were carried out at Kyungpook National University, Korea. UV−vis spectra were acquired on a Shimadzu UV−vis spectrophotometer (UV1650PC). Analytical HPLC traces were acquired using a Waters 600 series HPLC system and Waters Grace smart RP C18 column (4.6 × 250 mm, 5 μm), with the mobile phase consisting of 0.1% TFA/H2O (solvent A) and 0.1% TFA/acetonitrile (solvent B) and a gradient consisting of 1% B to 70% B in 20 min with a 1 mL/min flow rate. The radio-TLC measurements were performed using a Bioscan 2000 imaging scanner (Bioscan, Washington, DC, USA). S y nt h e s i s o f D i e t hy l - 2 - [ (4 - n i t r op h e ny l ) m e t h yl ] propanedioate (9). 4-Nitrobenzyl bromide, (10.57 g, 48.93 mmol) was added to a stirred solution of diethyl malonate, (52 mL, 54.86 g, 342.51 mmol) and K2CO3 (14.21 g, 102.8 mmol) in acetone (40 mL). The reaction mixture heated at 45 °C for 1 h. The mixture was allowed to cool to room temperature, and the resulting yellow slurry was filtered off to remove K2CO3 and washed with acetone (2 × 20 mL). The solvent was evaporated from the combined filtrate and wash liquid under reduced pressure. The excess diethylmalonate was removed by horizontal distillation (2 h, 80−130 °C and 0.005 mbar). The yellow oily residue was dissolved in ethanol (200 mL). The precipitated white solid (dialkylated product) was filtered and washed with ethanol (2 × 20 mL). The solvent was evaporated from the combined filtrate and wash material under reduced pressure to provide a solid, which was recrystallized from hexane/Et2O (1:1) to afford white crystalline needles of 9 (12.72 g, 88% yield). 1H NMR (500 MHz, CDCl3): δ 1.20−1.23 (t, 6H, J = 7 Hz), 3.31−3.32 (d, 2H, J = 7.5 Hz), 3.65−3.68 7240

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Journal of Medicinal Chemistry

Article

Synthesis of 4,11-N,N′-Bis(carboxymethyl)-16-(4-nitrobenzyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane·2TFA (11· 2TFA) (6). Compound 5 (2.56 g, 4.24 mmol) was dissolved in a 1:1 (vol/vol) mixture of CF3CO2H (TFA) and CH2Cl2 (70 mL). The mixture was stirred at room temperature for 24 h. The solvent was removed under reduced pressure to yield an oily residue, which was triturated with Et2O to provide the off-white solid of 6 as a TFA salt (2.93 g, 96% yield). 1H NMR (500 MHz, D2O): δ 8.12−8.10 (dd, 2H, J = 8.5 Hz), 7.47−7.45 (dd, 2H, J = 8.5 Hz), 3.92−3.22 (m, 12H), 3.20−2.52 (m, 12H), 2.48−2.05 (m, 5H), 1.79 (br s, 1H), 1.53 (br s, 1H). 13C NMR (125.8 MHz, D2O): δ 174.07, 169.66, 162.36 (q, JCF = 35.7 Hz, CF3COOH), 146.64, 146.51, 130.15, 123.98, 113.01 (q, JCF = 292.3 Hz, CF3COOH), 60.46, 59.44, 56.79, 55.68, 53.43, 51.50, 48.99, 47.68, 36.14, 31.64, 22.78, 16.70. HRMS (FAB) calculated for C24H38N5O6, 492.2822 [(M + H)+]; found, 492.2819 [(M + H)+]. Synthesis of 4,11-N,N′-Bis(carboxymethyl)-16-(4-aminobenzyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane·2TFA (7·2TFA) (7). To a solution of compound 6 (4.26 g, 5.92 mmol) in water (50 mL) was added 10% Pd/C (1.28 g). The resulting mixture was stirred under H2 (g) at room temperature for 12 h. The reaction mixture was filtered through celite pad, which was washed with water (2 × 20 mL). The combined filtrate was evaporated under vacuum to generate an oily residue, which was triturated with Et2O to provide the off-white solid of compound 7 (4.01 g, 98% yield). 1H NMR (500 MHz, D2O): δ 7.36−7.35 (dd, 2H, J = 8.5 Hz), 7.27−7.26 (dd, 2H, J = 8 Hz), 4.10−2.51 (m, 27 H), 2.50−1.51 (m, 6H). 13C NMR (125.8 MHz, D2O): δ 173.69, 169.14, 162.2 (q, JCF = 34.9 Hz, CF3COOH), 139.09, 130.34, 128.19, 122.99, 112.74 (q, JCF = 289.1 Hz, CF3COOH), 60.09, 59.11, 56.27, 55.17, 52.58, 51.24, 48.63, 47.27, 35.31, 30.95, 22.45, 16.18. HRMS (FAB) calculated for C24H40N5O4, 462.3080 [(M + H)+]; found, 462.3085 [(M + H)+]. Synthesis of 4,11-N,N′-Bis(carboxymethyl)-16-(4-isothiocyanatobenzyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane (8). To a solution of compound 7 (1.21 g, 1.75 mmol) in 0.5 M HCl (50 mL) was carefully added thiophosgene (CSCl2, CAUTION!) (4.03 mL, 6.04 g, 52.5 mmol) in CHCl3 (50 mL). The reaction mixture was stirred for 3 h at room temperature, and the layers were allowed to separate. The aqueous layer was removed, and the organic CHCl3 layer was then washed with water (2 × 50 mL). The combined aqueous layers were washed with CHCl3 (3 × 50 mL) to remove the unreacted thiophosgene. Finally, the aqueous layer was lyophilized to afford the white solid of 8 (0.86 g, 97% yield). 1H NMR (500 MHz, D2O): δ 7.30 (s, 2H), 7.07(s, 2H), 4.01−2.81 (m, 18 H), 2.80−1.89 (m, 12 H), 1.29 (br s, 1H). 13C NMR (125 MHz, DMSO-d6): δ 173.81, 168.70, 138.26, 134.23, 130.54, 128.80, 125.97, 60.26, 59.05, 55.56, 55.03, 52.58, 51.32, 48.45, 47.52, 35.56, 31.06, 22.88, 16.25. HRMS (FAB) calculated for C25H38N5O4S, 504.2645 [(M + H)+]; found, 504.2647 [(M + H)+]. Synthesis of Cu-PCB-TE2A-NH2. To a solution of PCB-TE2ANH2 7 (156 mg, 0.34 mmol) and Cu(ClO4)2·6H2O (126 mg, 0.34 mmol) in 20 mL of methanol was added 1 M aqueous solution of NaOH (2.41 mL, 2.41 mmol). The resulting clear blue solution was refluxed for 2 h, cooled, and filtered through a celite bed. The filtrate was subjected to diethyl ether diffusion. The deposited blue crystals were collected and dried (155 mg, 88% yield). HRMS (FAB): calculated for C24H38CuN5O4, 523.2220 [(M + H)+]; found, 523.2219 [(M + H)+]. Elemental analysis: calculated for C24H38CuN5O4·3H2O, C 47.94, H 7.50, N 12.71; found, C 47.37, H 6.83, N 11.26. Visible electronic spectrum: λmax(H2O)/635 nm; λmax(5 M HCl)/651 nm. Acid Decomplexation Studies by HPLC. The sample concentration of the copper complex (Cu-PCB-TE2A-NH2) studied was 3 mmol in 12 M HCl. The UV HPLC spectrum in 12 M HCl at 90 °C was recorded at specific time points by injecting an aliquot (20 μL) onto a reverse-phase Xbridge C18 column (4.6 × 150 mm, 5 μm) using an isocratic method (0.1% TFA/H2O−MeOH 91−9, and 1 mL/ min flow rate). The decreasing absorbance in the UV region (280 nm) was used to monitor the progress of the decomplexation reaction. Electrochemical Studies. Cyclic voltammetry was conducted with a Biologic model SP-150 with a three-electrode configuration. The working electrodes were a glassy carbon (diameter = 3 mm), Ag/

AgCl (sat. KCl) reference electrode and Pt wire counter electrode. The samples (1 mM) were run in 0.2 M phosphate buffer adjusted to pH 7.0 with glacial acetic acid at a scan rate of 100 mV/s. The solutions were deoxygenated for 15 min with argon prior to use and kept under argon atmosphere during measurement. Conjugation of PCB-TE2A-NCS to RGD Peptide. A solution of PCB-TE2A-NCS (8.61 μmol, 4.33 mg) was mixed with c(RGDyK) (2.87 μmol, 1.78 mg) in 0.1 M Na2CO3 buffer (pH 9.5). After stirring at room temperature overnight in the dark, the PCB-TE2A-NCS conjugated to c(RGDyK) peptide was isolated by semipreparative HPLC [Waters μbondapak C18; 10 μm, 7.8 × 300 mm; flow rate 3 mL/min, with the mobile phase starting from 95% solvent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in acetonitrile) (0−2 min) to 35% solvent A and 65% solvent B at 32 min]. The peak containing the PCB-TE2A-NCS-c(RGDyK) conjugate was collected at a retention time of 17.2 min. The collected fraction was combined and lyophilized to afford the final product as a white powder. PCB-TE2ANCS-c(RGDyK) was obtained in 72% yield with 15.7 min retention time on analytical HPLC [Grace smart RP C18; 5 μm, 4.6 × 250 mm; flow rate 1 mL/min, with the mobile phase consisting of 0.1% TFA/ H2O (solvent A) and 0.1% TFA/acetonitrile (solvent B), and a gradient consisting of 1% B to 70% B in 20 min]. The purified PCBTE2A-NCS-c(RGDyK) compound was identified by electrospray mass spectrometry (m/z calculated for C52H79N14O12S, 1122.56; found m/z, 1122.49 for [M]−). 64 Cu Radiolabeling of PCB-TE2A-NCS-c(RGDyK). 64Cu (0.5−2 mCi) in 100 μL of 0.1 M NaOAc buffer (pH 8.0) was added to 5 μg of PCB-TE2A-NCS-c(RGDyK) in 100 μL of 0.1 M NaOAc buffer (pH 8.0). The reaction mixture was incubated at 80 °C for 1 h. The reaction was monitored by radio-TLC using Whatman MKC18F TLC plates developed with 30:70 10% NH4OAc/methanol (64Cu-PCBTE2ANCS-c(RGDyK), Rf = 0.9). The 64Cu-labeled peptide was further purified by reverse-phase HPLC (RP-HPLC) using a Grace smart RP C18 column (5 μm, 4.6 × 250 mm) eluted with a mobile phase consisting of 0.1% TFA/H2O (solvent A) and 0.1% TFA/ acetonitrile (solvent B), and a gradient consisting of 1% B to 70% B in 20 min at a flow rate of 1 mL/min. 64Cu-PCB-TE2A-NCS-c(RGDyK) (retention time [tR] 17.1 min) was collected in 1−2 mL of HPLC solvent. The radiochemical purity of the radiolabeled peptide was measured using the same HPLC conditions as above. Conjugation and Radiolabeling of ECB-TE2A-c(RGDyK). ECB-TE2A was conjugated with c(RGDyK) peptide by using standard NHS activation method. First, cyclic(RGDyK) peptide was synthesized following typical solid phase synthesis (Fmoc protection). Then, ECB-TE2A (3.4 mg, 9.9 × 10−6 mole) and c(RGDyK) (3.4 mg, 5.5 × 10−6 mole) were dissolved in anhydrous DMSO. To this solution, 2 M N,N′-diisopropylcarbodiimide (7.5 μL) and of 2 M N,N′-diisopropyl ethylamine (20 μL) was added. Resulted mixture was stirred at room temperature for 15 h. The crude reaction mixture was purified by preparative HPLC to afford pure ECB-TE2A-c(RGDyK), which was characterized by electrospray mass spectrometry (m/z calculated for C43H70N13O11, 944.53; found m/z, 944.8 for [M + H]+). 64 Cu-labeled ECB-TE2A-c(RGDyK) was prepared by following a published procedure.29 Briefly, 10 μg of ECB-TE2A-c(RGDyK) was incubated with 64CuCl2 (1−2 mCi) in 0.1 M NH4OAc buffer (pH 8.0) at 95 °C for 30 min. The radiotracer was purified by HPLC by following similar conditions as those described for 64Cu-PCB-TE2ANCS-c(RGDyK). Radiochemical purity was >90% before HPLC purification and was over 99% after HPLC purification. Specific Activity Measurement. For specific activity measurement, a nonradioactive Cu-PCB-TE2A-NCS-c(RGDyK) was prepared by incubating PCB-TE2A-NCS-c(RGDyK) with excess CuCl2 (10 mol equivalent) at 90 °C for 1 h. The pH of the reaction mixture was adjusted to 8.0 by 0.1 M NaOH. Various amount of Cu-PCB-TE2ANCS-c(RGDyK) was injected into HPLC, and a calibration plot was prepared for area under the peak for Cu-PCB-TE2A-NCS-c(RGDyK) at 220 nm wavelength versus known amount of Cu-PCB-TE2A-NCSRGDyK injected. Peak area obtained in UV chromatogram at 220 nm for the purified 64Cu-PCB-TE2A-NCS-c(RGDyK) was used to calculate specific activity (μCi/μg). 7241

dx.doi.org/10.1021/jm500348z | J. Med. Chem. 2014, 57, 7234−7243

Journal of Medicinal Chemistry

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Determination of Partition Coefficient. The logP values of Cu-PCB-TE2A-NCS-c(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK) were determined by adding 5 μL of the labeled tracer (∼50 μCi) to a mixture of 500 μL of 1-octanol and 500 μL of water. The resulting solutions were vigorously vortexed for 5 min at room temperature, then centrifuged for 5 min to ensure complete separation of layers. From each of the six sets, a 100 μL aliquot was removed from each phase into screw tubes and counted separately in a gamma counter. The partition coefficient was calculated as a ratio of counts in the 1octanol fraction to counts in the water fraction. The logP values were reported in an average of six measurements. This experiment was carried out in duplicate. Integrin Receptor Binding Assay. The cell-binding assay of the PCB-TE2A-NCS-c(RGDyK) was compared with c(RGDyK) (reference molecule) using 125I-echistatin on U87MG cells as described previously.33 U87MG cells (2 × 106/100 μL) were resuspended in binding buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MnCl2, and 0.1% bovine serum albumin). For the assay, equal volumes of nonradioactive ligand PCB-TE2A-NCS-c(RGDyK) and radioactive ligand (0.1 μCi 125I-echistatin, PerkinElmer, Branford, CT) were added. Increased concentrations (10−5 to 10−14 M) of the ligands were added to each tube. The tubes were incubated for 60 min at room temperature, and then the reaction medium was removed and the cells were washed three times with PBS. The cells were harvested, and the bound 125I-echistatin was counted in a gamma counter, 1480 WIZARD (Wallac). Data was analyzed with GraphPad Prism 5 (GraphPad softwere, Inc., San Diego, CA) to determine the IC50 value. Experiments were carried out with triplicate samples. Animal Models. All animal experiments were conducted in compliance with the Animal Care and Use Committee requirements of Kyungpook National University. Xenograft tumor models of U87MG cell lines were prepared using 6 week-old BALB/c nu/nu female nude mice. A total of 5 × 106 U87MG cells were inoculated subcutaneously into the right flank of the mice. Tumors of appropriate size usually grew within 15 d after the tumor cell implantation. In Vivo Stability. The in vivo stability of 64Cu-PCB-TE2A-NCSc(RGDyK) was evaluated by following reported processes.27,28 For direct comparison purposes, in vivo stability of 64Cu-ECB-TE2Ac(RGDyK) was also evaluated in the same method. Approximately 1 mCi of radiolabeled conjugates (64Cu-PCB-TE2A-NCS-c(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK)) were injected intravenously in Sprague−Dawley rats. The blood was collected from the heart at 1 h postinjection and immediately centrifuged for 10 min at 13,000 rpm. To the supernatant (200 μL), a 100 μL mixture of acetonitrile−water− trifluoroacetic acid (50:45:5) was added, followed by mixing, keeping at 4 °C for 10 min and centrifugation for 10 min at 13,000 rpm. The supernatant was then filtered through a 0.22 μm filter, and the filtrate was injected to reverse-phase HPLC [Grace smart RP C18 column (5 μm, 4.6 × 250 mm) eluted with a mobile phase consisting of 0.1% TFA/H2O (solvent A) and 0.1% TFA/acetonitrile (solvent B), and a gradient consisting of 1% B to 70% B in 20 min at a flow rate of 1 mL/ min]. The eluent was collected with a fraction collector (1.0 mL/ fraction), and the radioactivity of each fraction was measured with a gamma counter. For the liver and kidney, samples were first homogenized well with homogenizer, and then 5 mL of PBS was added and vortexed for 5 min followed by centrifugation for 10 min at 13,000 rpm. To the supernatant (200 μL), a 100 μL mixture of acetonitrile−water− trifluoroacetic acid (50:45:5) was added, followed by mixing, keeping at 4 °C for 10 min and centrifugation for 10 min at 13,000 rpm. The supernatant was then filtered through a 0.22 μm filter, and the filtrate was injected to reverse-phase HPLC; collected fractions were measured with a gamma counter as per previously described for blood sample. Biodistribution of 64Cu-PCB-TE2A-NCS-c(RGDyK). Tumorbearing BALB/c nude mice (n = 3) were injected via tail-vein with 64 Cu-PCB-TE2A-NCS-c(RGDyK) (ca. 20 μCi in 150 μL saline per mice). The animals were sacrificed at 1, 4, and 24 h postinjection. The organs and tissues of interest (blood, heart, muscle, fat, bone, spleen, kidney, intestine, liver, and tumor) were collected, weighted, and

analyzed using a gamma counter. The percent of injected dose per gram (%ID/g) was calculated by comparison to a weighted, counted standard. The receptor-mediated localization of the radiotracer (blocking study) was investigated by injection of nonradioactive c(RGDyK) peptide at 5 mg/kg along with 64Cu-PCB-TE2A-NCSc(RGDyK) in the same tumor model, and biodistribution was carried out at 1 h postinjection. MicroPET Imaging in U87MG Tumor Bearing Nude Mice. Small animal PET scans and image analysis were performed using an Inveon PET/CT scanner (Siemens). The imaging studies were carried out on female nude mice bearing xenograft U87MG tumors on their right flank. The mice were injected via the tail vein with 64Cu-PCBTE2A-NCS-cRGDyK (∼500 μCi). At 1, 4, and 24 h after injection, the mice were anesthetized with 1% to 2% isoflurane, positioned in the prone position, and imaged. The images were reconstructed by a 2dimensional ordered-subsets expectation maximum (OSEM) algorithm, and no corrections were necessary for attenuation or scatter. An ROI was placed on the liver, kidney, muscle, and tumor in the coronal microPET images for quantification purpose.

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ASSOCIATED CONTENT

* Supporting Information S

NMR (1H and 13C) and HR-MS spectra of all synthesized compounds, HR-MS of Cu-PCB-TE2A-NH2, ESI mass spectra of PCB-TE2A-NCS-c(RGDyK) and ECB-TE2A-c(RGDyK), and biodistribution data of 64Cu-PCB-TE2A-NCS-c(RGDyK). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(J.Y.) E-mail: [email protected]. Author Contributions

∥ These authors (D.N.P., N.B., and G.I.A.) contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an R&D program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2013R1A2A2A01012250, 2013M2A2A6042317, and 2014032805), and the Basic Research Laboratory (BRL) Program (2013R1A4A1069507). The Korea Basic Science Institute (Daegu) is acknowledged for providing assistance with the NMR and MS measurements.

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ABBREVIATIONS USED BFCs, bifunctional chelators; DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid; TETA, 1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N‴-tetraacetic acid; NOTA, 1,4,7triazacyclododecane-N,N′,N″-triacetic acid; DTPA, diethylene triamine pentaac etic acid; ECB-TE2 A, 4, 11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6,6,2]hexadecane; PCB-TE2A, 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6,6,3]hexadecane; NCS, isothiocyanate; EtOAc, ethyl acetate; Et2O, diethyl ether; c(RGDyK), cyclic Arg-Gly-Asp-DTyr-Lys peptide; FBS, fetal bovine serum; U87MG, Human glioblastoma-astrocytoma epithelial-like cell line; KIRAMS, Korea Institute of Radiological and Medical Sciences; tR, retention time in chromatography 7242

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