Synthesis, Copper(II) Complexation, 64Cu-Labeling, and

Bioconjugate Chem. 2008, 19, 719–730

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Synthesis, Copper(II) Complexation, 64Cu-Labeling, and Bioconjugation of a New Bis(2-pyridylmethyl) Derivative of 1,4,7-Triazacyclononane Gilles Gasser,†,‡ Linda Tjioe,† Bim Graham,*,§ Matthew J. Belousoff,† Stefanie Juran,‡ Martin Walther,‡ Jens-Uwe Künstler,‡ Ralf Bergmann,‡ Holger Stephan,*,‡ and Leone Spiccia*,† School of Chemistry, Monash University, Clayton, Vic 3800, Australia, Forschungszentrum Dresden-Rossendorf, Institute of Radiopharmacy, PF 510119, D-01314 Dresden, Germany, and Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, Parkville, Vic 3052, Australia. Received October 25, 2007; Revised Manuscript Received November 16, 2007

A new ligand derivative of 1,4,7-triazacyclononane (TACN), 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan1-yl]acetic acid (6), has been synthesized and its complexation behavior toward Cu2+ ions investigated. The ligand 6 has been characterized by spectroscopic methods, and a molecular structure of a corresponding Cu(II) complex has been elucidated by single-crystal X-ray analysis. The suitability of 6 for conjugation to peptide substrates has been shown by amide coupling of 6 to the stabilized derivative of bombesin (BN), βAla-βAla[Cha13, Nle14]BN(7–14), to give the conjugate 8. The free ligand 6 and the bioconjugate 8 were labeled with 64 Cu2+, and the resulting complexes, 64Cu⊂6 and 64Cu⊂8, were found to be stable in the presence of a large excess of a competing ligand (cyclam) or copper-seeking superoxide dismutase (SOD), as well as in rat plasma. Biodistribution studies of 64Cu⊂8 in Wistar rats showed a high activity uptake into the pancreas (5.76 ( 0.25 SUV, 5 min p.i.; 3.93 ( 0.25 SUV, 1 h p.i.), which is the organ with high levels of gastrin-releasing peptide receptor (GRPR). This receptor is overexpressed in a large number of breast and prostate carcinomas. The novel 64 Cu⊂6 complex had a dominating influence on the nonspecific activity biodistribution of its BN conjugate, since the distribution data of 64Cu⊂6 are similar to those of 64Cu⊂8. The 64Cu complexes exhibited a low activity accumulation in the liver tissue and an extensive renal clearance, which was distinctively different to the biodistribution of 64CuCl2, suggesting that 64Cu⊂6 does not undergo significant demetalation, but rather exhibits high in ViVo stability.

INTRODUCTION The selection of potentially useful copper radionuclides in nuclear medicine is wide and includes 60Cu, 61Cu, 62Cu, 64Cu, and 67Cu (1–6). However, due to their favorable decay characteristics and the ability to produce them with high specific activity on a biomedical cyclotron, 64Cu (t1/2 ) 12.7 h; β+max ) 0.653 MeV, 17.4%; β-max ) 0.578 MeV, 39%) and 67Cu (t1/2 ) 61.8 h; β-max ) 0.576 MeV, 100%) are the preferred copper isotopes for radiopharmaceutical applications. Incorporation of transition metal radionuclides, such as 64Cu and 67Cu, into target-specific radiopharmaceuticals requires the use of chelating ligands to bind the radionuclide to the conjugate (7–9). An extremely important requirement is that the resulting radionuclide-ligand complex is both kinetically and thermodynamically stable in ViVo in order to minimize release of the isotope to normal tissue, thus reducing the background for imaging and minimizing radiation exposure to normal tissue for therapy (10). A large variety of amine-based polydentate chelating agents have been investigated in an effort to satisfy this requirement, such as polyaminocarboxylates (11–14), cyclic polyamines (azamacrocycles) (15–17), cyclic polyaminocarboxylates (1, 15) and pyridine-containing polyamines (18–22). In general, azamacrocyclic ligands are preferred over acyclic ligands, as the structural features of the macrocyclic Cu(II) complexes render them more kinetically stable under biological * Corresponding authors. Email: [email protected], Fax: +61 3 9903 9582. Email: [email protected], Fax: +49 351 260 3232. Email: [email protected], Fax: +61 3 9905 4597. † School of Chemistry, Monash University. ‡ Forschungszentrum Dresden-Rossendorf, Institute of Radiopharmacy. § Victorian College of Pharmacy, Monash University.

conditions (14, 16, 23–25). Macrocyclic tetraamines such as 12-N4 (1,4,7,10-tetraazacyclododecane, “cyclen”), 13-N4, and 14-N4 (1,4,8,11-tetraazacyclotetradecane, “cyclam”) form very stable complexes with Cu(II) and are resistant to demetalation in ViVo (14, 16). Furthermore, their carboxylate-containing analogues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-1,4,8,11tetraacetic acid (TETA), can be coupled to biomolecules. Notably, a copper-based radiopharmaceutical featuring the macrocyclic ligand TETA as a chelating agent has advanced to clinical trials (26, 27). Unfortunately, serious problems associated with retention of radioactivity in blood, liver, and bone narrow were found for this bioconjugate (64Cu-TETA-Octreotide), suggesting that dissociation of Cu2+ ions from the radiopharmaceutical, and subsequent binding of the ions to proteins such as superoxide dismutase (SOD) in rat livers, is a problem even for this macrocycle (28). 64Cu complexes of macrobicyclic tetraaminocarboxylates have shown remarkably improved in ViVo stability, but harsh labeling conditions has hampered their application (29–32). Another approach to achieve highly stable Cu(II) complexes is through the use of multidentate ligands containing pyridine donor groups, such as N,N′,N′′-tris(2-pyridylmethyl)-cis,cis1,3,5-triaminocyclohexane (tachpyr), 3,7-diazabicyclo[3.3.1]nonane (bispidine), and azamacrocycles with pendant pyridyl groups (10, 18–20, 22, 33, 34). The latter two ligand structures, in particular, allow the convenient introduction of additional carboxylic groups, enabling their coupling to appropriate biomolecules. Thus, it should be possible to develop new pyridine-containing chelating agents that are able to rapidly form stable radiocopper complexes for targeted imaging and therapeutic purposes. In an effort to address this challenge, we have

10.1021/bc700396e CCC: $40.75  2008 American Chemical Society Published on Web 02/07/2008

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Gasser et al.

Scheme 1. Synthesis of 6a

Figure 1. Structure of bioconjugate 8.

a (a) (i) 2-Picolyl chloride, THF, r.t., o/n; (ii) H2O, reflux, 4 h, 79%; (b) picolyl chloride hydrochloride, K2CO3, KI, CH3CN, reflux, o/n, 77%; (c) 4 M HCl(aq), reflux, 4 h, 85%; (d) Ethylbromoacetate, K2CO3, KI, CH3CN, reflux, o/n, 88%; (f) LiOH.H2O, ethanol/H2O, 70°C, o/n, 85%.

embarked on a program to develop improved copper-based radiopharmaceuticals featuring pyridyl derivatives of the tridentate macrocycle 1,4,7-triazacyclononane (TACN) as the chelating agent. Due to its small ring size, TACN coordinates facially to metal ions such as Cu2+, with the metal lying out of the plane defined by the three nitrogen atoms. The donor atoms are oriented in such a way as to maximize orbital overlap and thereby produce complexes with very high thermodynamic stabilities (35). Herein, we report the synthesis and copper coordination chemistry of the TACN derivative, 2-[4,7-bis(2-pyridylmethyl)1,4,7-triazacyclononan-1-yl]acetic acid (6) (Scheme 1), that contains two pyridyl pendant groups to increase the stability of the copper complex significantly compared to the parent TACN macrocycle and a carboxylic acid functionality that allows the covalent attachment of 6 to tumor-specific biomolecules via an amide coupling reaction. This possibility for regiospecific coupling is a convincing advantage compared to other previously described pyridine-containing polyamine ligands (36). To demonstrate the utility of 6 in preparing 64Cu-labeled radiotracers, we chose in the first instance to conjugate it to bombesin (BN), a tetradecapeptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR). The GRPR is overexpressed on a variety of tumors, including commonly diagnosed breast and prostate cancers, and the less common pancreatic and small-cell lung cancers (37–39). To date, research investigations have examined BN derivatives radiolabeled mainly with 99mTc, but also to a lesser extent with 64Cu, 18F, 68 Ga, 125I, 111In, 177Lu, 188Re, 86Y, and 90Y (38–40). 64Cu conjugates with DOTA-PEG-BN(7–14) (PEG ) poly(ethylene glycol)) (41), DOTA-[Lys3]BN (42, 43), DOTA-X-BN(7–14) (X ) spacer containing 4, 5, 6, 8, or 12 carbon atoms) (43–46), DOTA-X-BN(7–14) (X ) tripeptide spacer Gly-Gly-Gly, GlySer-Gly, Gly-Ser-Ser, Gly-Glu-Gly, or Gly-Glu-Glu) (47), and DOTA-[Pro1, Tyr4]BN (40) have been described, as well as 64Cu conjugates with CB-TE2A-8-Aoc-BN(7–14) (CB-TE2A, 1,4,8,11tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid; 8-Aoc, 8-aminooctanoic acid) (46), or NOTA-X-BN(7–14) (NOTA, 1,4,7triazacyclononane-1,4,7-triacetic acid; X ) single amino acid spacer containing 3, 5, or 8 carbon atoms, Gly-Gly-Gly or SerSer-Ser) (48). The bombesin analogue selected in this work was βAla-βAla[Cha13, Nle14]BN(7–14) () βAla-βAla-Gln-Tpr-Ala-Val-GlyHis-Cha-Nle-NH2), whose 99mTc-tricarbonyl conjugate (BBS-

38) was investigated by García Garayoa et al. (49, 50). This BN analogue is characterized by the replacement of the C-terminal natural amino acids Leu13 and Met14 by Cha13, Nle14, and the further addition of a βAla-βAla spacer group. The Tctricarbonyl conjugate of this analogue exhibited an increased metabolic stability in blood plasma and tumor cells, as well as positively affecting the tumor-to-blood/kidney/liver ratio in comparison to the nonstabilized BN(7–14), without a spacer or with other spacers (49, 50). With this in mind, we report herein the conjugation of the TACN derivative 6 to βAla-βAla-[Cha13, Nle14]BN(7–14), resulting in the preparation of the bioconjugate 8 (Figure 1) and present the results of subsequent 64Cu labeling experiments with 6 and 8 to give 64Cu⊂6 and 64Cu⊂8, respectively. In Vitro and in ViVo stability investigations, as well as biodistribution studies, are also presented and discussed.

EXPERIMENTAL SECTION Materials. All solvents used were of reagent or analytical grade and used as purchased. CH3CN was dried by standing over activated 4 Å molecular sieves overnight. Distilled water was used for all reactions in aqueous solution. CO2-free water was prepared by boiling distilled water under nitrogen for 2 h and cooling while bubbling with nitrogen gas. High-purity nitrogen gas was used directly from a reticulated system. βAlaβAla-Gln-Trp-Ala-Val-Gly-His-Cha-Nle-NH2 (βAla-βAla-[Cha13, Nle14]BN(7–14)) was obtained from Biosyntan GmbH, Germany. Rat blood plasma was prepared from Wistar rats (Harlan Winkelmann GmbH, Borchen, Germany). 64Cu was produced on the PET cyclotron “Cyclone 18/9” of the FZ DresdenRossendorf by the 64Ni(p,n) f 64Cu nuclear reaction according to the procedure reported previously (51, 52). Instrumentation and Methods. Infrared spectra were recorded as NaCl plates, nujol, or neat liquids as noted on a Perkin-Elmer 1600 series FTIR spectrophotometer at a resolution of 4.0 cm–1. Diamond ATR spectra were recorded on a Specac “Golden Gate” apparatus. Abbreviations used to describe the peak intensities of IR and ATR spectra are as follows: vs (very strong), s (strong), m (medium), w (weak), sh (shoulder), and br (broad). Microanalytical (C, H, and N) analyses were performed by Campbell Microanalytical Service, Otago, New Zealand. 1H NMR and 13C NMR spectra were measured on a Bruker AC200, DPX300, or DRX400 spectrometer. Tetramethylsilane (TMS) was used as an internal calibrant for spectra recorded in CDCl3 solutions. Abbreviations used to describe the resonances for 1H NMR spectra are as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), or br (broad singlet). Low-resolution electrospray mass spectra were obtained with a Micromass Platform II Quadrupole Mass Spectrometer fitted with an electrospray source. High-resolution mass spectra were recorded on a Bruker BioApex II 47e FT-ICR MS fitted with an analytical elctrospray source. Samples were introduced by syringe pump at a flow rate of 1 µL/min. The capillary voltage was 200 V. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on an Autoflex TOF/TOF instrument from Bruker Daltonics, Germany. HPLC was performed on a Perkin-Elmer device consisting of a Turbo LC System with a quaternary pump

Bis(2-pyridylmethyl) Derivative of 1,4,7-Triazacyclononane

(series 200 LC Pump), a programmable absorbance detector model 785A (Perkin-Elmer Instruments GmbH, Germany), and a homemade γ-ray detector (well-type, NaI(T1) crystal). Eluent A, 0.1% (v/v) TFA in CH3CN; eluent B, 0.1% (v/v) TFA in H2O; eluent C, 0.05% (v/v) TFA in CH3CN; eluent D, 0.04% (v/v) TFA in H2O. HPLC system 1, Jupiter (Phenomenex) 4 µ Proteo 90Å, 250 × 4.6 mm; gradient elution, 10% to 70% of eluent A in 40 min, 1 mL/min. HPLC system 2, Jupiter (Phenomenex) 4 µ Proteo 90Å, 250 × 10 mm, gradient elution, 10% to 70% of eluent A in 40 min, 3 mL/min. HPLC system 3, Zorbax (Agilent) SB-C18, 300 Å, 5 µm, 250 × 9.4 mm; gradient elution using eluents C and D, 10% to 70% eluent C in 20 min, 3 mL/min, 28 °C. Thin layer chromatography (TLC) was performed using basic alumina (Al2O3) plates with detection of species present by KMnO4 oxidation (eluents specified in the synthesis section). TLC of the 64Cu-labeled compounds was performed using neutral alumina plates which were recorded on a Radioisotope Thin Layer Analyzer (Rita Star, Raytest Isotopenmessgerät GmbH, Straubenhardt, Germany). The eluent was a 1:1 mixture by volume methanol/ammonium acetate (5% in water) [Rf ) 0 (Cu2+); Rf ) 1 (64Cu⊂6)]. 1,4,7-Triazacyclo[5.2.1.04,10]decane (1). 1 was synthesized following the previously reported procedure. Analytical data were in agreement with that described in the literature (53, 54). 1-Formyl-4-(2-pyridylmethyl)-1,4,7-triazacyclononane (2). Picolyl chloride hydrochloride was neutralized with an excess of NaOH, and extraction with DCM yielded neutral picolyl chloride. A sample of this compound (6.02 g, 0.0472 mol) was dissolved in THF (50 mL) and then added dropwise to a solution of 1 (6.35 g, 0.050 mol) in THF (50 mL). The resulting mixture was stirred overnight at room temperature. The orange precipitate which formed was filtered off, washed with THF, and dried under reduced pressure. The solid was dissolved in water (50 mL) and the solution refluxed for 4 h. The solution was cooled to room temperature and aqueous 5 M NaOH added dropwise until a pH of 13 was reached. The compound was then extracted with CHCl3, the organic phase dried with anhydrous MgSO4, filtered, and the solvent removed under vacuum to give 2 as a yellow oil. Yield: 9.21 g (79%). 1H NMR (300 MHz, CDCl3): δ 2.50 (s br, 1H, NH), 2.60–2.70 (m, 3H, CH2TACN ring), 2.85 (m, 1H, CH2TACN ring), 3.01 (m, 1H, CH2TACN ring), 3.10–3.25 (m, 4H, CH2 TACN ring), 3.40–3.50 (m, 3H, CH2 TACN ring), 3.88 (s, 2H, N-CH2-pyridyl), 7.18 (m, 1H, CH pyridyl), 7.38 (m, 1H, CH pyridyl), 7.68 (m, 1H, CH pyridyl), 8.12 (s, 1H, CHO), 8.54 (m, 1H, CH pyridyl). 13C NMR (50.29 MHz, CDCl3): 46.5, 46.6, 47.0, 47.6, 47.9, 49.1, 49.8, 51.7, 52.4, 53.2, 55.1, 56.5 (all CH2 TACN ring), 62.9 (N-CH2pyridyl), 121.8, 121.9, 122.7, 122.8, 136.1, 136.3, 136.4, 148.8 (all CH pyridyl), 159.3 (C pyridyl), 159.4 (C pyridyl), 163.4 (CHO), 163.5 (CHO). IR (KBr disk, cm-1): 3369w (νN-H), 3049s (νC-H(Aromatic)), 2923br (νC-H), 1662s (νC)O), 1590s (νC)N(Pyridyl)), 1569s (νC)C(Pyridyl)), 1435s br (νC)C(Pyridyl)), 1365s br, 1273s, 1150s, 1047s, 993s, 733s, 700s. ESI-MS electrospray mass spectrum (m/z): 249.2 (100%) [M + H]+, 261.2 (10%) [M + Na]+. High-resolution ESI mass determination (MeOH): Found, 249.1708; calcd for C13H21N4O, 249.1715. 1-Formyl-4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononane (3). A solution of picolyl chloride hydrochloride (6.12 g, 0.0371 mol) in CH3CN (50 mL) was added dropwise to a mixture of 2 (9.21 g, 0.0371 mol) in CH3CN (50 mL) containing K2CO3 (20.50 g, 0.1480 mol) and KI (0.10 g, 0.6020 mmol). The resulting mixture was stirred for 1 h at room temperature and then refluxed overnight. The reaction mixture was cooled to room temperature, filtered, and washed with acetonitrile. The filtrate was evaporated under reduced pressure to yield 3 as a brown oil. Yield: 9.69 g (77%). 1H NMR (300 MHz, CDCl3): δ 2.59 (m, 2H, CH2TACN ring), 2.81 (m, 2H,

Bioconjugate Chem., Vol. 19, No. 3, 2008 721

CH2TACN ring), 3.03 (m, 2H, CH2TACN ring), 3.27 (m, 4H, CH2TACN ring), 3.42 (m, 2H, CH2TACN ring), 3.87 (s, 2H, N-CH2-pyridyl), 3.88 (s, 2H, N-CH2-pyridyl), 7.14 (m, 2H, CH pyridyl), 7.43 (m, 2H, CH pyridyl), 7.66 (m, 2H, CH pyridyl), 8.05 (s, 1H, CHO), 8.53 (m, 2H, CH pyridyl). 13C NMR (50.29 MHz, CDCl3): 46.5, 47.0, 50.2, 53.0, 54.2, 57.7 (all CH2 TACN ring), 62.9 (N-CH2-pyridyl), 63.6 (N-CH2-pyridyl), 120.9, 121.8, 122.5, 122.8, 135.6, 135.8, 148.8, 148.9 (all CH pyridyl), 159.6 (C pyridyl), 159.8 (C pyridyl), 163.5 (CHO). IR (KBr disk, cm-1): 3052s (νC-H(Aromatic)), 3008s (νC-H), 2926s br(νC-H), 2824s br, 2250s, 1666s (νC)O), 1589s (νC)N(Pyridyl)), 1569s (νC)C(Pyridyl)), 1473s (νC)C(Pyridyl)), 1435s (νC)C(Pyridyl)), 1371s br, 1314s, 1225s, 1148s, 1047s, 994s, 757s. ESI-MS electrospray mass spectrum (m/z): 340.1 (100%) [M + H]+. High-resolution ESI mass determination (MeOH): Found, 340.2133; calcd for C19H26N5O, 340.2137. 1,4-Bis(2-pyridylmethyl)-1,4,7-triazacyclononane (4). 3 (9.69 g, 0.0280 mol) was dissolved in aqueous 4 M HCl (50 mL) and the resulting solution refluxed for 4 h. The solution was then allowed to cool to room temperature, and aqueous 5 M NaOH (50 mL) was added dropwise until a pH of 13 was reached. The compound was extracted with CHCl3 (3 × 50 mL), the organic phase washed with anhydrous MgSO4, filtered, and the solvent removed under vacuum to yield 4 as a reddish brown oil. Yield: 7.55 g (85%). Anal. Found (%): C, 69.32; H, 7.76; N, 22.42. Calcd for C18H25N5 (%): C, 69.48; H, 7.65; N, 22.32. 1 H NMR (400 MHz, CDCl3): δ 2.75 (m, 4H, CH2TACN ring), 2.95 (m, 4H, CH2TACN ring), 3.06 (m, 4H, CH2TACN ring), 3.89 (s, 4H, N-CH2-pyridyl), 4.71 (s br, 1H, NH), 7.14 (m, 2H, CH pyridyl), 7.26 (m, 2H, CH pyridyl), 7.58 (m, 2H, CH pyridyl), 8.61 (m, 2H, CH pyridyl). 13C NMR (75.44 MHz, CDCl3): 45.4 (CH2 TACN ring), 49.7 (CH2 TACN ring), 52.8 (CH2 TACN ring), 61.02 (N-CH2-pyridyl), 121.7, 122.4, 122.7, 123.1, 136.2, 136.6, 148.8, 149.3 (all CH pyridyl), 158.3 (C pyridyl). IR (KBr disk, cm-1): 3416w (νN-H), 3052s (νC-H(Aromatic)), 2919s br (νC-H), 1590s (νC)N(Pyridyl)), 1568s (νC)C(Pyridyl)), 1472s (νC)C(Pyridyl)), 1434s (νC)C(Pyridyl)), 1362s, 1148s, 995s, 752s, 662s. ESI-MS Electrospray mass spectrum (m/z): 312.3 (100%) [M + H]+. Ethyl-2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan1-yl]acetate (5). A solution of ethyl bromoacetate (0.50 g, 2.99 mmol) in dry CH3CN (25 mL) was added dropwise to a mixture of 4 (0.92 g, 3.0 mmol) in dry CH3CN (25 mL) containing K2CO3 (1.64 g, 11.8 mmol) and KI (0.10 g, 0.60 mmol). The resulting mixture was stirred for 1 h at room temperature, and then refluxed overnight. It was subsequently cooled to room temperature, filtered, and the residue washed with dry acetonitrile. The filtrate was collected, and the solvent removed under reduced pressure to yield 5 as a brown oil. Yield: 1.18 g (88%). 1 H NMR (200 MHz, CDCl3): δ 1.28 (t, 3J ) 7.1 Hz, 3H, CH3CH2O), 2.45–2.85 (m, 12H, CH2TACN ring), 3.35 (s, 2H, N-CH2-COO), 3.86 (s, 4H, N-CH2-pyridyl), 4.12 (q, 3J ) 7.1 Hz, 2H, CH3-CH2O), 7.19 (m, 2H, CH pyridyl), 7.46 (m, 2H, CH pyridyl), 7.62 (m, 2H, CH pyridyl), 8.54 (m, 2H, CH pyridyl). 13C NMR (75.44 MHz, CDCl3): δ 13.1 (OCH2-CH3), 53.0 (b, CH2 TACN ring), 59.0 (N-CH2-COO), 59.2 (OCH2CH3), 63.5 (N-CH2-pyridyl), 120.9 (CH pyridyl), 121.9 (CH pyridyl), 134.7 (CH pyridyl), 147.6 (CH pyridyl), 158.1 (C pyridyl), 170.8 (COO). IR (KBr disk, cm-1): 3050s (νC-H(Aromatic)), 2930s br (νC-H(TACN)), 1732s (νC)O), 1592s (νC)N(Pyridyl)), 1568s (νC)C(Pyridyl)), 1472s (νC)C(Pyridyl)), 1435s (νC)C(Pyridyl)), 1372w, 1198w, 1049w, 994s br, 761s. ESI-MS Electrospray mass spectrum (m/z): 398.3 (100%) [M + H]+. 2-[4,7-Bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic Acid (6). To 5 (1.18 g, 3.0 mmol) dissolved in ethanol (30 mL) was added LiOH · H2O (0.45 g, 10.7 mmol) dissolved in water (10 mL). This mixture was stirred at 70 °C overnight. It


was then cooled to room temperature, and the pH was adjusted to 7 by addition of aqueous 1 M HCl. The solvent was removed under reduced pressure and dichloromethane used to extract the solid. The solution was filtered and the filtrate dried with Na2SO4. After filtration, the solvent was removed under reduced pressure to yield 6 as a brown oil. Yield: 1.02 g (85%). Anal. Found (%): C, 65.18; H, 7.22; N, 18.88. Calcd for C20H27N5O2 (%): C, 65.09; H, 7.38; N, 18.98. 1H NMR (75.44 MHz, CDCl3): δ 2.40–2.80 (m, 12H, CH2TACN ring), 3.19 (s, 2H, N-CH2COOH), 3.89 (d, 2J ) 15.2 Hz, 2H, N-CH2-pyridyl), 3.98 (d, 2 J ) 15.2 Hz, 2H, N-CH2- pyridyl), 7.14 (m, 2H, CH pyridyl), 7.28 (m, 2H, CH pyridyl), 7.72 (m, 2H, CH pyridyl), 8.17 (m, 2H, CH pyridyl). 13C NMR (75.44 MHz, CDCl3): δ 49.9 (CH2 TACN ring), 50.0 (CH2 TACN ring), 50.3 (CH2 TACN ring), 59.3 (N-CH2-COOH), 60.5 (N-CH2-pyridyl), 122.1 (CH pyridyl), 122.3 (CH pyridyl), 136.9 (CH pyridyl), 148.6 (CH pyridyl), 157.5 (C pyridyl), 176.1 (COOH). IR (KBr disk, cm-1): 3390w br (νO-H), 3059w (νC-H(Aromatic)), 2939m (νC-H), 2867s (νC-H), 2826s, 2359s, 2192s, 1667s (νC)O), 1599s (νC)N(Pyridyl)), 1571s (νC)C(Pyridyl)), 1481s (νC)C(Pyridyl)), 1456s (νC)C(Pyridyl)), 1436s (νC)C(Pyridyl)), 1365s (νC-O), 1149s, 1119s, 1097s, 1046s, 1005s, 978s, 914m, 836s, 727m, 641s. ESI-MS Electrospray mass spectrum (m/z): 368.3 (100%) [M - H]-. Ethyl-2-{2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan1-yl]acetamido}acetate (7). 6 (0.65 g, 1.8 mmol) was suspended in acetonitrile (15 mL), and HBTU (0.67 g, 1.8 mmol), DIPEA (1.60 g, 12.4 mmol), and glycine ethyl ester hydrochloride (0.25 g, 1.78 mmol) added. The brown reaction mixture was stirred overnight at room temperature, and thereafter the solvent was removed under reduced pressure. To the residue was added dichloromethane (10 mL), and the mixture was washed with saturated NaHCO3 solution (5 mL) and water (2 × 5 mL). After drying of the organic phase over Na2SO4 and subsequent filtration, the solvent was removed under reduced pressure. The crude product was purified by column chromatography on basic alumina with CH2Cl2/MeOH (98:2 by volume) as the eluent (Rf ) 0.28) to yield 7 as a yellow oil. Yield: 0.32 g (40%). Anal. Found (%): C, 63.55; H, 7.46; N, 18.65. Calcd for C24H34N6O3 (%): C, 63.49; H, 7.55; N, 18.52. 1H NMR (75.44 MHz, CDCl3): δ 1.19 (t, 3J ) 7.1 Hz, 3H, CH3-CH2O), 2.40–2.80 (m, 12H, CH2TACN ring), 3.40 (s, 2H, N-CH2CONH), 3.87 (d, 2J ) 15.3 Hz, 2H, N-CH2-pyridyl), 3.98 (d, 2 J ) 15.3 Hz, 2H, N-CH2-pyridyl), 4.02 (d, 3J ) 5.8 Hz, 2H, CONH-CH2-COO), 4.12 (q, 3J ) 7.1 Hz, 2H, OCH2-CH3), 7.02 (t, 3J ) 5.8 Hz, 1H, CONH), 7.19 (m, 4H, CH pyridyl), 7.71 (m, 2H, CH pyridyl), 8.07 (m, 2H, CH pyridyl). 13C NMR (300 MHz, CDCl3): δ 14.1 (OCH2-CH3), 41.6 (CONH-CH2-COO), 51.1 (br, CH2 TACN ring), 51.3 (CH2 TACN ring), 59.6 (NCH2-CONH), 61.3 (N-CH2-pyridyl), 61.7 (COO-CH2-CH3), 122.9 (CH pyridyl), 123.1 (CH pyridyl), 137.8 (CH pyridyl), 149.1 (CH pyridyl), 158.4 (C pyridyl), 169.3 (CH2-COO-CH2), 173.6 (CONH). IR (KBr disk, cm-1): 3406w (νN-H), 3035w (νCH(Aromatic)), 2953s (νC-H), 2867s (νC-H), 2826s, 2359s, 1789s (νC)O(ester)), 1658s (νC)O(amide)), 1599s (νC)N(Pyridyl)), 1571s (νC)C(Pyridyl)), 1481s (νC)C(Pyridyl)), 1456s (νC)C(Pyridyl)), 1436s (νC)C(Pyridyl)), 1365s (νC-O), 1149s, 1097s, 1046s, 978s, 914m, 836s, 727m, 641s. ESI-MS Electrospray mass spectrum (m/z): 455.3 (100%) [M + H]+. Bioconjugate 6-βAla-βAla-[Cha13, Nle14]BN(7–14) (8). 6 (2.3 mg, 6.2 µmol), HBTU (4.7 mg, 12 µmol) and βAla-βAlaGln-Trp-Ala-Val-Gly His-Cha-Nle-NH2 (5.0 mg, 4 µmol) were dissolved in DMF (2 mL). DIPEA (5.0 µL, 29 µmol) was then added and the mixture stirred for 20 h at room temperature. The DMF was removed under high vacuum and the residual brown oil was purified HPLC (system 2) to yield 8 as a white solid after lyophilization. Yield: 3.6 mg (61%). Electrospray mass spectrum (m/z): 728 [M + 2H]2+, 739 [M + H + Na]2+,

Gasser et al.

1456 [M + H]+, 1478 [M + Na]+. MALDI-TOF MS: Found: 1457.1; calculated for C73H107N20O12: 1455.8. HPLC system 1: tR ) 19.2 min. [Cu(6-H)Cu(NO3)2.MeOH]NO3.MeOH(Cu2⊂6).Cu(NO3)2 · 3H2O (59 mg, 0.24 mmol) was dissolved in MeOH (5 mL) and added to a solution of 6 (89 mg, 0.24 mmol) in MeOH (5 mL). The color changed to dark green and a brown precipitate formed. The solution was filtered to remove the precipitate. Slow diffusion of a ether into this solution produced crystals that were suitable for X-ray crystallography. Yield: 16 mg (18%). Anal. Found (%): C, 37.60; H, 4.43; N, 15.05. Calcd for Cu2C22H34N8O13 (%): C, 35.43; H, 4.50; N, 15.02. IR (ATR, cm-1): 3049w (νC-H(Aromatic)), 2932s (νC-H), 2862s (νC-H), 1610s (νC)O), 1573s (νC)C(Pyridyl)), 1440s (νC)C(Pyridyl)), 1330s br (νNO3-), 1021m, 976m, 826m, 777m. X-ray Crystallography. Intensity data for a blue crystal of Cu2⊂6 (0.21 × 0.19 × 0.06 mm3) was measured at 123 K on a Bruker Apex 2 CCD fitted with graphite-monochromated Mo KR radiation (0.71073 Å). The data were collected to a maximum 2θ value of 60° and processed using the Bruker Apex 2 software. Crystal parameters and details of the data collection for Cu2⊂6 are summarized and presented in the Supporting Information. The structure was solved by direct methods and expanded using standard Fourier routines in the SHELX-97 (55, 56) software package. All hydrogens except for H1M and H2M, which were located on the Fourier-difference map, were placed in idealized positions, and all nonhydrogen atoms were refined anisotropically. Preparation of 64Cu⊂6 and 64Cu⊂8. An aqueous solution of 64CuCl2 (50 MBq, 100 µL, 0.1 M NH4OAc) was added to ca. 1 mg of 6 or 8 dissolved in 100 µL MeCN/H2O (1:1 by volume). The reaction mixtures were tempered at 40 °C for 30 min, which results in 90% labeling yield. The crude products were then purified by HPLC (system 2). This results in a radiochemical purity >97%. For in ViVo studies, the obtained fraction was concentrated to dryness and redissolved in 0.9% aqueous saline solution; tR ) 4.4 min for 64Cu⊂6, tR ) 19.5 min for 64Cu⊂8 (HPLC system 1). Concentration Dependence. To 100 µL aqueous solution of ligand 6 (10-2 M, 10-3 M, 10-4 M, and 10-5 M in 0.1 M NH4OAc), 200 kBq of 64CuCl2 (100 µL in 0.1 M NH4OAc) was added. The mixtures were shaken for 10 min and then analyzed by TLC. Kinetic Studies. 100 µL of an aqueous solution of ligand 6 (10-4 M in 0.1 M NH4OAc) was spiked with 64CuCl2 (100 kBq) and stirred. Aliquots of the mixtures were analyzed by TLC to determine the degree of complexation. Samples were taken after 1, 5, 10, and 30 min. Ligand “Challenge” Experiment. A solution containing 100 kBq 64CuCl2 was added to 500 µg of 6 dissolved in 100 µL of 0.1 M NH4OAc. After 10 min, full complexation of the ligand was checked by TLC. Cyclam (40 mg, more than 100-fold excess) dissolved in 1 mL 0.1 M NH4OAc was then added to the complex solution, which was stirred for 24 h. Aliquots of the mixtures were assayed by TLC and HPLC (system 1) to determine the degree of decomposition. SOD “Challenge” Experiment. A solution of 250 kBq 64 CuCl2 in 0.1 M NH4OAc was added to 10 µg of 6 dissolved in 100 µL acetonitrile/water (1:5). The full complexation of the ligand was checked via TLC after 15 min. After the removal of acetonitrile, 1.35 mg of SOD (from bovine liver) was dissolved in 200 µL of 0.9 M NaCl solution-representing a more than 1000-fold excess-and added to the prepared complex solution, which was then stirred for 24 h. The degree of decomposition was determined by TLC using aliquots of the mixtures.


Determination of Partition Coefficient log Do/w. Ligands 6 or 8 dissolved in 50 µL water (c0 )10-3 M) were added to a mixture consisting of 390 µL HEPES/NaOH buffer (0.05 M, pH ) 7.2, 7.4, 7.8), a 10 µL 64CuCl2 solution in 0.1 M NH4OAc, and 50 µL of a 10-4 M aqueous solution of Cu(NO3)2. The distribution experiments in 1-octanol/buffer system were carried out at 25 ( 1 °C in microcentrifuge tubes (2 cm3) with mechanical shaking. The phase ratio V(1-octanol):V(aq) was 1:1 (0.5 cm3 each). The shaking time was 30 min. All samples were centrifuged and the phases separated. The copper concentration in both phases was determined radiometrically using γ-radiation [64Cu, NaI(Tl) scintillation counter automatic gamma counter 1480, Wizard 3′′, PerkinElmer]. The results are the average values of three independent experiments. In Vitro Stability. 64Cu⊂6 or the bioconjugate 64Cu⊂8 were incubated in phosphate buffer (250 µL, Sörensen, pH ) 7.4) and rat plasma (250 µL) at 37 °C for 2 h and then at 25 ( 1 °C for 24 h. Cold ethanol (500 µL) was added to the mixture, and after centrifugation for 5 min, the ethanol phase was removed from the protein precipitate. This procedure was repeated another time. The ethanol solution was dried under nitrogen, and a solution of H2O/CH3CN (9:1 by volume) (100 µL) was added and acidified with TFA (5 µL). Samples containing 64Cu⊂6 were analyzed using TLC and HPLC (system 1); 64Cu⊂8 samples analyzed only by HPLC. The whole experiment was repeated two times. In ViWo Stability. The in ViVo stability of 64Cu⊂8 was analyzed using rat arterial blood samples at various time points (1, 3, 5, 10, 20, 30, and 60 min) after injection of the radiotracer. The samples were centrifuged to precipitate the blood cells. A solution consisting of 50 µL CH3CN, 45 µL H2O, and 5 µL TFA was added to 200 µL of the supernatant, and the mixture was then cooled to -20 °C. After centrifugation, the supernatant was analyzed by HPLC (system 3). The chromatogram of 64 Cu⊂8 (plasma, 0 min) was obtained by addition of 64Cu⊂8 to rat blood and immediately treating the mixture as described for the in ViVo samples. Urine and kidney homogenates were treated analogously to the arterial blood samples. Animals, Feeding, Husbandry and Biodistribution Studies in Rats. The animal research committee of the Regierungspräsidium Dresden approved the animal facilities and the experiments according to institutional guidelines and the German animal welfare regulations. The Wistar rats (Harlan Winkelmann GmbH, Borchen, Germany) were housed under standard conditions with free access to standard food and tap water. Animals were between 7 and 9 weeks of age. The rats were housed in an Animal Biosafety Level 1 (ABSL-1) acclimatized facility with a temperature of 22 ( 2 °C and humidity of 55 ( 5%. Animals were under a 12 h light cycle in temperature (27 ( 1 °C) controlled-airflow cabinets. The animals had free access to standard pellet feed and water. Four animals (body weight 227 ( 10 g) for each time point were intravenously injected into a tail vein with approximately 8 µCi (0.3 MBq) 64Cu⊂8 conjugate in 0.5 mL electrolyte solution E-153 (Serumwerk Bernburg, Germany) with 5% Tween 80. Animals were euthanized at 5 and 60 min postinjection. Blood and the major organs were collected, weighed, and counted in a Wallac WIZARD Automatic Gamma Counter (PerkinElmer, Germany). The radioactivity of the tissue samples was decay-corrected and calibrated by comparing the counts in tissue with the counts in aliquots of the injected tracer that had been measured in the gamma counter at the same time. The activity in the selected tissues and organs was expressed as percent injected dose per organ (% ID) or as standardized uptake value (SUV). Values are quoted as means ( standard deviation (mean ( SD) for each group of four animals.


PET Imaging. General anesthesia of rats was induced with inhalation of desflurane 9% (v/v) (Suprane, Baxter, Germany) in 40% oxygen/air (gas flow 1 L/min), and was maintained with desflurane 6% (v/v). For arterial blood sampling, a 3.5 Fr umbilical vessel catheter prefilled with heparinized saline was inserted into the right carotid arteria, and 100 µL blood samples were taken at 1, 3, 5, 10, 20, 30, and 60 min after tracer application. In the PET experiments, 0.1 mCi (3.7 MBq) of the 64 Cu⊂8 conjugate in 0.5 mL was administered intravenously over 1 min into a tail vein. PET imaging of 64Cu⊂8 in rat was performed over 60 min with a microPET P4 scanner (Siemens CTI Molecular Imaging Inc., Knoxville). Data acquisition was performed in 3D list mode. A transmission scan was carried out prior to the injection of 64Cu⊂8 using a 137Cs point source. Emission data were collected continuously for 60 min after injection of 64Cu⊂8. The list mode data were sorted into sinograms using a framing scheme of 12 × 10 s, 6 × 30 s, 5 × 300 s, and 5 × 600 s frames. The data were attenuation corrected, and the frames were reconstructed by Ordered Subset Expectation Maximization applied to 3D sinograms (OSEM3D) with 14 subsets, 6 OSEM3D iterations, 2 maximum a posteriori (MAP) iterations, and 0.05 beta-value for smoothing. The pixel size was 0.8 by 0.8 by 1.2 mm3, and the resolution in the center of field of view was 1.85 mm. No correction for recovery and partial volume effects was applied. The image files were processed using the ASIPro software (Siemens, CTI Molecular Imaging Inc. Knoxville) and the ROVER software (ABX GmbH, Radeberg, Germany). Summed frames from 30 to 90 s postinjection (p.i.) and 30 to 90 min p.i. were used to define the regions of interest (ROI). The data were normalized to the injected radioactivity by using 18F standards from the injection solution measured in a γ-well counter (Isomed 2000, Dresden, Germany) cross-calibrated to the PET scanner and expressed in SUV. Statistical Analysis. The data are expressed as means ( SD. One-way analysis of variance (ANOVA) was used for statistical evaluation. Means were compared using Student’s t test. A P value of 10-4 M was found to be necessary to

Bioconjugate Chem., Vol. 19, No. 3, 2008 725 Table 1. Partition Coefficients (log Do/w) of the and 8 at Different pHs

64

Cu Complexes of 6

pH

log Do/w (64Cu⊂6)

log Do/w (64Cu⊂8)

7.2 7.4 7.8

-2.43 -2.45 -2.43

-2.39 -2.38 -2.36

obtain complete copper complexation. At a ligand concentration of approximately 10-5 M, 10% of free Cu2+ ions were still present in solution after 10 min. The rate of copper(II) complexation with 6 was found to be very fast in ammonium acetate buffer solution (60) at ambient temperature, viz., the TACN derivative 6 (10-4 M) was successfully labeled with 64Cu within 1 min in quantitative yield. Lipophilicity. Information about the lipophilicity of the copper complex 64Cu⊂6 and the bioconjugate 64Cu⊂8 was obtained by measuring their partitioning in a 1-octanol/water system. The partition coefficients (log Do/w) were measured at different pH in buffered solution. The results are summarized in Table 1. The copper complexes of both the ligand 6 and the bioconjugate 8 are hydrophilic. This indicates that they should be predestined to show rapid blood clearance and preferential renal excretion. Interestingly, the log Do/w values of the 64Cu⊂8 complex remain unchanged in the pH range investigated, indicating that the protonation state of the peptide does not influence the value of log Do/w. In Vitro Stability. Competition experiments in the presence of the cyclam macrocycle, a ligand that forms one of the most stable Cu(II) complexes known (14), were undertaken to check the stability of the 64Cu complexes. 64Cu⊂6 and64Cu⊂8 were exposed to more than a 100-fold excess of cyclam. No transchelation was observed after 24 h, using HPLC as the detection system [tR ) 4.4 min (Cu⊂6), tR ) 5.3 min (Cu⊂cyclam), tR ) 19.2 min (Cu⊂8), HPLC system 1; see Experimental Section]. The in Vitro stability of 64Cu⊂6 and 64 Cu⊂8 was also assessed in a rat plasma medium after 2 and 24 h. The 64Cu⊂6 complex remained unchanged. In the case of 64Cu⊂8, after 2 h, another small peak was detected. It was found that this peak was not due to free copper in solution, resulting from dissociation of copper(II) ions from the chelate complex, but instead arose from cleavage of the peptide of 64 Cu⊂8, presumably brought about by the action of peptidases. Competition experiments were also performed in the presence of SOD (Cu/Zn superoxide dismutase). SOD is a 32 kDa enzyme that is distributed in the cytosol of eukaryotic cells and is particularly abundant in liver, kidney, and red blood cells. This enzyme plays a key role as a copper-binding protein (28, 30). Ligand 6 was labeled with 64Cu and spiked with a 1000-fold excess of SOD. After 24 h, the stability of the 64Cu⊂6 complex (Rf ) 1) was checked via TLC. We could not detect the presence of 64Cu⊂SOD (Rf ) 0), which was synthesized for comparison as previously described (28). Thus, the absence of protein-bound radioactivity indicates no transchelation of copper(II). Overall, the results of the competition studies showed that both 64Cu⊂6 and 64Cu⊂8 are very stable. This finding is in accordance with our preliminary potentiometric measurements, which indicate that the stability constant for the copper complex is >1025 M-1. In ViWo Stability. The metabolic stability of 64Cu⊂8 was determined in arterial blood samples taken at 1, 3, 5, 10, 20, 30, and 60 min after a single intravenous application of the radiotracer in a rat. The blood samples were centrifuged to obtain the plasma and then treated with acetonitrile to precipitate the proteins. The obtained supernatant was referred to as the plasma extract. The mean relative activity in the plasma was 89 ( 8% of arterial blood activity; the activity of the plasma extract samples contained 66 ( 13% of the plasma activity. The plasma extract was analyzed by RP-HPLC for radiometabolites (Figure


Figure 4. Examples of radiochromatograms (HPLC system 3) of 64 Cu⊂8 and rat plasma extract, kidney extract, and urine at various time points after single intravenous application of 64Cu⊂8.

4). The relative amount of the original compound 64Cu⊂8 (tR ) 12.9 min) decreased from 93% at the time of tracer application (1 min 77%, 3 min 60%, 5 min 50%, 10 min 31%, 20 min 14%, 30 min 6%) to 3% of the total plasma extract activity after one hour (Figure 4). 64Cu⊂8 was converted into

Gasser et al.

three more hydrophilic radiolabeled compounds (tR ) 3.7, 5.3, 9.1 min). The peak at 3.7 min was not caused by 64Cu2+ since it was eluted at 3.4 min. The residual activity in the kidney extract resulted from only one hydrophilic species (tR ) 3.7 min). The activity of the urine was caused by the same three species detected in the plasma extract. The original compound 64 Cu⊂8 could not be detected in the kidney extract or urine. This extensive metabolism of the compound could be speciesspecific for the rat. Biodistribution Studies. A summary of the biodistribution and elimination data for the bombesin conjugate 64Cu⊂8, 64 Cu⊂6, and 64CuCl2 in rats is shown in Figures 5 and 6. The values of %ID, %ID/g, and SUV (ID ) injected dose; SUV ) standardized uptake value) are summarized in the Supporting Information. The SUV was used to normalize the biodistribution data and calculated as followed: SUV ) tissue activity concentration (Bq/g) × body weight (g)/injected activity (Bq) (injected and tissue activity were decay-corrected). A high uptake of 64Cu⊂8 was observed in the GRPR-expressing pancreas (5.8 ( 0.7 [SUV] at 5 min after injection, and 3.9 ( 0.3 [SUV] after 1 h). After one hour, the activity concentration was highest in the pancreas of all the organs examined. In contrast to these findings, the activity concentration in the pancreas after injection of 64Cu⊂6 and 64CuCl2 was very low, and roughly comparable to the blood activity uptake. The 64 Cu⊂8 had a rapid blood clearance, with 2.0 ( 0.3 (SUV) in the circulation at 5 min, followed by a further decrease to 0.23 ( 0.04 (SUV) at 1 h. Liver uptake reached 6.3 ( 0.7% ID at 5 min and declined to 3.7 ( 0.3% ID one hour after injection. The activity detected in the intestine attained 5.3 ( 0.4% ID at 5 min and 9.9 ( 2.4% ID after one hour. In the kidneys, the activity attained 5.3 ( 0.5% ID at 5 min and decreased to 1.6 ( 0.1% ID at one hour. Most activity was eliminated into the urine (13 ( 7% ID at 5 min and 57 ( 4% ID at 1 h). A very similar activity distribution and excretion pattern was observed

Figure 5. Biodistribution of 64Cu⊂8, 64Cu⊂6, and 64CuCl2 in male Wistar rats 5 and 60 min after single intravenous application represented as %ID mean ( SD (4 animals per time point). (%ID, %ID/g, and SUV data are given in the Supporting Information).



Figure 6. Biodistribution of 64Cu⊂8, 64Cu⊂6, and 64CuCl2 in male Wistar rats 5 and 60 min after single intravenous application, represented as SUV mean ( SD (4 animals per time point). (%ID, %ID/g, and SUV data are given in the Supporting Information).

Figure 7. Small animal coronal (A), sagittal (B), and transversal (C) PET images of rat 60 min after single intravenous application of 64Cu⊂8 into a rat tail vein.

after injection of 64Cu⊂6, which exemplifies the strong influence of the novel 64Cu-TACN chelate to the biodistribution of its bombesin conjugate. In contrast, the activity distribution of 64 CuCl2 was distinctively different and characterized by a high liver uptake and a very low renal clearance. From the different biodistribution characteristics, it can be deduced that there was only marginal, or no, in ViVo demetalation of the 64Cu⊂6 unit. The remarkable kidney uptake of 64Cu⊂8 in comparison to the relatively low liver accumulation is also demonstrated by the small PET images (Figure 7) (note that the pancreas is hard to see due to its proximity to the duodenum of the small intestine). A variety of radiolabeled bombesin derivatives, or analogues, containing the BN(8–14) sequence have been described in the literature, which show varying pharmacokinetic behavior depending on the radiolabeling approach taken, the spacer between the radiometal chelate, and the peptide N-terminus, and the specific modifications made to the peptide sequence. Since peptides are rapidly metabolized; bombesin derivatives with increased stability toward peptidases have been designed to tackle shortcomings, such as the intermediate or low target uptakes, and high liver and/or kidney uptakes, observed for most of the radiolabeled bombesin conjugates reported so far. Stabilized radiometal-labeled bombesin derivatives include [DPhe6,βAla12,Nle14]BN(6–14)(61),[Cha13]BN(7–14)(49,50,61), [Nle14]BN(7–14) (49, 50, 61, 62), [Cha13, Nle14]BN(7–14) (49, 50), [Pro1, Tyr4]BN (40), [Pro1, Tyr4, Nle14]BN (62), [DPhe6, Leu-

NHEt13, des-Met14]BN(6–14) (63, 64), and [DTyr6, βAla11, Thi13, Nle14]BN(6–14) (64–66), with the N-terminus conjugated to the radiometal complexes mainly via spacers. The bombesin used in the present work was selected from a study on 99mTctricarbonyl labeled bombesin derivatives in which the radiotracer 99m Tc-BBS-38 was found to show an improved target/nontarget ratio due to the spacer βAla-βAla that stabilized BN(7–14) via Cha13 and Nle14 (49, 50). The uptake level of 64Cu⊂8 in the pancreas at 1 h p.i. was roughly comparable to the values for 99mTc-BBS-38 at 1.5 h p.i. The uptake of 64Cu⊂8 in the kidneys and the lungs was significantly higher compared to 99mTc-BBS-38. The rapid decrease of activity accumulation in the kidneys and the increasing activity in the urine suggest a predominant renal clearance pathway for 64Cu⊂8, in contrast to the dominant hepatobiliary elimination of 99mTc-BBS-38 (49). The pancreatic uptake of 64Cu⊂8 at 1 h p.i. was roughly in the same range as that described for 64Cu-labeled DOTA bombesin conjugates studied in mice (41, 42, 44–48). 64Cu-labeled DOTA-PEGBN(7–14) (41), DOTA-X-BN(7–14) (X ) spacer containing 4, 5, 6, 8, or 12 carbon atoms) (43–46, 48) and DOTA-XBN(7–14) (X ) Gly-Gly-Gly, Gly-Ser-Gly, or Gly-Ser-Ser) (47) resulted in a relatively high liver uptake, which was higher, or nearly the same, as the activity uptake into the kidneys. In contrast, 64Cu⊂8 exhibited a lower uptake in the liver compared to the kidneys. 64Cu-DOTA-[Lys3]BN (42, 43), 64Cu-DOTA-


[Pro1, Tyr4]BN (40), 64Cu-CB-TE2A-8-Aoc-BN(7–14) (46), and 64 Cu-NOTA-8-Aoc-BN(7–14) (48) were characterized by a predominant renal excretion pathway comparable to 64Cu⊂8, which showed an even lower liver uptake than 64Cu-DOTA[Lys3]BN (42, 43) at 21 h p.i.

CONCLUSION An efficient synthetic strategy has been developed for the preparation of a new TACN derivative, 2-[4,7-bis(2-pyridylmethyl)1,4,7-triazacyclononan-1-yl]acetic acid (6), containing two pyridine pendant arms as well as a carboxylic group for coupling to biomolecules. The X-ray structure of a copper complex of 6 confirmed that the ligand is capable of multidentate coordination to a Cu2+ ion. Conjugation of glycine ethyl ester and the bombesin analogue βAla-βAla-[Cha13, Nle14]BN(7–14) to 6 was successfully achieved via amide coupling. Both ligand 6 and the bombesin bioconjugate 8 can readily form radiocopper complexes, 64Cu⊂6 and 64Cu⊂8, with high stability. In Vitro ligand competition experiments and stability studies in rat plasma medium gave no evidence of transchelation or demetalation. The 64Cu complexes of 6 and 8 are relatively hydrophilic (log Do/w < -2.3). Biodistribution studies of the bombesin conjugate 64Cu⊂8 revealed an accumulation of the compound in the GRPR-expressing pancreas that is roughly comparable to the same bombesin analogue labeled with 99mTc (99mTc-BBS-38) (49). The bombesin conjugate 64Cu⊂8 showed an extensive renal clearance of the activity, in contrast to 99m Tc-BBS-38 or other BN(7–14) analogues labeled via 64CuDOTA (41, 43–48). The 64Cu-TACN unit had a dominating influence on the nonspecific activity biodistribution, since the distribution data of 64Cu⊂6 were very similar to that of 64Cu⊂8. Comparison with biodistribution data for 64CuCl2 indicated that there was only marginal, if any, in ViVo copper demetalation. The specificity of binding to the GRPR, as well as tumor uptake, will be evaluated in further experiments, and results will be reported in due course. Our radiopharmacological data support the potential of 64 Cu⊂8 for GRPR imaging and indicate that the chelating agent 6 may be an attractive candidate for developing new copper radiopharmaceuticals because it may be efficiently labeled under mild conditions (ambient temperature, aqueous solution) and exhibits fast renal clearance.

ACKNOWLEDGMENT This work was supported by the Swiss National Science Foundation and the Australian Research Council (ARC) through the Australian Centre for Electromaterials Science (ACES). G.G. was the recipient of a Swiss Fellowship for Prospective Researchers Grant (PBNE2-106771) and acknowledges financial support by the Forschungszentrum Dresden-Rossendorf during his stay in Germany. The authors thank Karin Landrock and Andrea Suhr for excellent technical assistance, and Christina Hultsch for MALDI-TOF measurements. Supporting Information Available: Crystallographic data for Cu2⊂6 is provided in cif format. Figure S1 shows the binuclear structure of Cu2⊂6. Tables of selected bond distances and angles for Cu2⊂6, of hydrogen bonds for Cu2⊂6 and of biodistribution of 64Cu⊂8, 64Cu⊂6, and 64CuCl2 in rats (mean ( SD of 4 animals per group) after single intravenous application in a tail vein (data in %ID, %ID/g, and SUV) are presented. This material is available free of charge via the Internet at http://pubs.acs.org.

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Synthesis, Copper(II) Complexation, 64Cu-Labeling, and

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