Hexadentate Bispidine Derivatives as Versatile Bifunctional Chelate

Bioconjugate Chem. 2009, 20, 347–359

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Hexadentate Bispidine Derivatives as Versatile Bifunctional Chelate Agents for Copper(II) Radioisotopes3 Stefanie Juran,† Martin Walther,† Holger Stephan,*,† Ralf Bergmann,† Jo¨rg Steinbach,† Werner Kraus,‡ Franziska Emmerling,‡ and Peter Comba*,§ Forschungszentrum Dresden-Rossendorf, Institute of Radiopharmacy, PF 510119, D-01314 Dresden, Germany, Bundesanstalt fu¨r Materialforschung and -pru¨fung, Richard-Willsta¨tter-Strasse 11, D-12489, Berlin, Germany, and Universita¨t Heidelberg, Anorganisch-Chemisches Institut, Im Neuenheimer Feld 270, D-69120 Heidelberg, Germany. Received October 24, 2008; Revised Manuscript Received December 22, 2008

The preparation and use of bispidine derivatives (3,7-diazabicyclo[3.3.1]nonane) as chelate ligands for radioactive copper isotopes for diagnosis (64Cu) or therapy (67Cu) are reported. Starting from the hexadentate bispidine-based bis(amine)tetrakis(pyridine) ligand 1 with a keto and two ester substituents, the corresponding mono-ol 2 and two dicarboxylic acid derivatives 3 and 5 have been synthesized. A range of techniques, including single-crystal X-ray structure analysis, UV/vis spectroscopy, cyclic voltammetry, thin-layer- (TLC), and high-performance liquid chromatography (HPLC), have been used to characterize the structure and stability of the copper(II)-bispidine complexes. A rapid formation (within 1 min) of stable copper(II)-bispidine complexes under mild conditions (ambient temperature, aqueous solution) has been observed. Challenge experiments of these complexes in the presence of a high excess of competing ligands, such as glutathione, cyclam, or superoxide dismutase (SOD), as well as in rat plasma, gave no evidence of demetalation or transchelation. The bifunctional bispidine derivative 5 can be readily functionalized with biologically active molecules at the pendant carboxylate groups. The coupling of a bombesin analogue βhomo-Glu-βAla-βAla-[Cha13,Nle14]BBN(7-14), by condensation of a carboxylate of the bispidine backbone with the N-terminus of the peptide produced the bifunctional ligand 6. The radiocopper(II) complex of this bombesin-bispidine conjugate has a considerable hydrophilicity (log Do/w < -2.4), and this leads to a very fast blood clearance (blood: 0.28 ( 0.02 SUV, 1 h p.i.), low liver tissue accumulation (liver: 1.20 ( 0.27 SUV, 1 h p.i.), and rapid renal-urinary excretion (kidneys: 6.06 ( 2.96 SUV, 1 h p.i.) as shown by biodistribution studies of 64Cu-6 in Wistar rats. Preliminary in vivo studies of 64Cu-6 in NMRI nu/nu mice, bearing the human prostate tumor PC-3 showed an accumulation of the conjugate in the tumor (2.25 ( 0.13 SUV, 12.5 min p.i.; 0.94 ( 0.05 SUV, 55 min p.i.) and allowed a clear visualization of the gastrin-releasing peptide receptor distribution by positron emission tomography (PET).

INTRODUCTION The extremely rigid diazaadamantane-derived bispidine ligands have first been prepared in an elegant, very versatile, and highyielding synthesis by Mannich and recently have found extensive applications in various areas of transition metal coordination chemistry (1-7). Similar to the alkaloid sparteine, from which the bispidines are derived, various derivatives are known for their analgetic and antiarrhythmic activity (1, 8, 9), and the stereochemistry of the substituents is of importance for the specific receptor binding (10-13). In the correct configuration, bispidines are highly preorganized and exceedingly rigid multidentate ligands leading to a number of very interesting properties of their transition metal coordination complexes (14-18). The distance of the ring nitrogen atoms N3-N7 of 2.9-3.1 Å allows a very stable chelation of first-row transition metals of medium size in different oxidation states, such as V4+/5+, Mn2+, Fe2+/3+, Co2+/3+, Ni2+, Zn2+, and preferably Cu2+. The number, type, and position * Corresponding authors. E-mail: [email protected], Fax: +49 351 260 3232; E-mail: [email protected], Fax: +49 6221 54 6617. 3 Dedicated to Professor Dr. Fritz Vögtle on the occasion of his 70th birthday. † Institute of Radiopharmacy. ‡ Bundesanstalt fu¨r Materialforschung and -pru¨fung. § Universita¨t Heidelberg.

of the donor groups, which can be varied during the bispidine synthesis, provides a variety of tailor-made coordination sites for metal ions with differing preferences with respect to size, shape, and electronic properties (1). In particular, hexadentate ligands with pyridine units in C2, C4, N3, and N7 positions have been shown to rapidly form stable copper(II) complexes under mild conditions. The stability constants of copper(II)bispidines are in the same range as those of macrocyclic ligand copper(II) complexes (1, 19, 20). As with other open-chained amine-pyridine based ligands (21-23), the relatively fast complexation kinetics of bispidine ligands in comparison with macrocyclic ligands makes the structurally reinforced bispidine derivatives with pendant pyridine donor groups attractive as chelators for copper(II) radioisotopes. Despite extensive work in this field, a number of challenges such as the specific coupling of ligands with targeting structures and detailed in vivo studies have not been accomplished to date. 64 Cu and 67Cu have suitable nuclear decay properties with a half-life of 12.7 and 62 h, respectively, and therefore offer the possibility to be established in tumor targeting for diagnosis and therapy (24-30). Coupling of the bispidine scaffold to targeting biomolecules is therefore of particular interest. Both the C9 and the C1/5 positions are potential centers to be used for the linkage to biomolecules. The conversion of the keto group to an alcohol at C9 (31) and/or hydrolyzation of the C1/5 ester groups (32) may provide linker groups for the coupling to vector molecules.

10.1021/bc800461e CCC: $40.75  2009 American Chemical Society Published on Web 01/27/2009

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Scheme 1. 64Cu-Labeled Bispidine-Bombesin Conjugate 6 (py ) pyridin-2-yl substituent)

Scheme 2. Synthetic Procedure for Preparation of the Reduced Derivative 2 and Ligand 3 with Two Free Carboxylic Groups at C1 and C5 (py ) pyridin-2-yl)a

a

(a) (i) NaBH4 in dioxane/water (1/1), r.t., 24 h, (ii) H2O, HCl, 55%; (b) CsOH, THF, r.t. 24 h, 66%.

Herein, we report the synthesis and copper coordination chemistry of the mono-ol 2 and the dicarboxylic acid derivatives 3 and 5, starting from the hexadentate bispidinebased bis(amine)tetrakis(pyridine) ligand 1 (Scheme 2). In this first exploratory study, a stabilized bombesin (BBN) derivative βhomoGlu-βAla-βAla-[Cha13,Nle14]BBN(7-14) (33) was attached as a biomolecule to the bispidine ligand 5, yielding the bispidine-bombesin conjugate 6 (Scheme 1) as a potential imaging probe for monitoring GRP receptor expression in vivo by means of PET. The radiopharmacological characterization of the 64Cu-labeled bispidine monool ester 64Cu-2, the dicarboxylic acid derivative 64Cu-3, and the bispidine-bombesin conjugate 64Cu-6 includes in vitro and in vivo metabolism and biodistribution studies in normal rats, and in small animal PET studies in NMRI nu/nu mice, bearing the human prostate tumor PC-3.

EXPERIMENTAL SECTION Materials. Chemical reagents and solvents were purchased from commercial sources and used without further purification. The enzyme superoxide dismutase (from bovine liver) was purchased from Sigma. The hexadentate bispidine ligand 1 has been prepared according to a literature procedure (34). βhomoGlu-βAla-βAla-[Cha13,Nle14]BBN(7-14) was obtained from Biosyntan GmbH, Berlin, Germany. The production of 64Cu was performed at a PET cyclotron, similar to those recently described for the 64Cu-61Co-production process (35). For the 64Ni(p,n)64Cu nuclear reaction, 15 MeV protons with a beam current of 10 µA for 2 h were used. The complete separation of 64Cu and 61 Co was confirmed by γ-ray spectroscopy. Nickel targets were prepared by electrodeposition of enriched 64Ni (99.6%) on Au disks at amounts of 30-120 mg. The plated diameter was 7 mm, matching more than 80% intensity of the Cyclone 18/9 proton beam, as measured by autoradiography of a natNi disk irradiated with 15 MeV protons to induce thenatNi(p,x)57Ni reaction. Production runs have yielded 1.5-6.0 GBq Cu-64 (130-331 MBq/µA/h) with specific activities of 30-200 GBq/ µmol Cu (35-37). Methods and Instrumentation. 1H and 13C NMR spectra were recorded on a 400 MHz Varian Inova spectrometer. The chemical shifts (δ, ppm) are relative to TMS or the solvent.

Elemental analyses were performed on a Leco Elemental Analyzer CHNS-932. Electrospray ionization mass spectrometry (ESI-MS) was carried out using a micromass tandem quadropole mass spectrometer (Quattro LC). High-resolution mass spectra were recorded on an Agilent 6210 ESI-TOF instrument, Agilent Technologies, Santa Clara, CA, USA (ESI-TOF ) electrospray ionization-time-of-flight); the solvent flow rate was adjusted to 4 µL/min; the spray voltage was set to 4 kV. The drying gas flow rate was set to 1 bar; all other parameters were adjusted to maximize [M + H]+. A Cary 50 Bio UV/vis spectrometer (Varian) was used to record electronic absorption spectra. Thin layer chromatography (TLC) was performed using neutral Al2O3 TLC plates (Merck, F254), developed in a 1:1 mixture methanol/ ammonium acetate (0.1 M in water). Radioactive detection of 64 Cu-labeled compounds was assayed on a radioisotope thin layer analyzer (Rita Star, Raytest Isotopenmessgera¨t GmbH, Straubenhardt, Germany). Electrochemical measurements were carried out with a BAS100B system with a standard threeelectrode cell (glassy carbon electrode, Ag/AgNO3 reference electrode (0.01 M AgNO3), Pt-wire auxiliary electrode) at 25 °C under argon atmosphere. All samples were measured in acetonitrile (0.1 M (Bu)4NPF6) or water (0.1 M KNO3) with a scan rate of 100 mV/s. HPLC was performed using either of two eluents (eluent A: acetonitrile, containing 0.1% trifluoroacetic acid; eluent B: water, containing 0.1% trifluoroacetic acid), three instruments, and five methods. HPLC system 1: Knauer Smartline, including a pump 1000, a UV detector 2500, and a manager 5000. Method 1: Jupiter 4 µm C18 90 Å (Phenomenex), 250 mm × 4.6 mm, elution gradient 10% to 70% A in 20 min, 1 mL/min. Method 2: Jupiter 4 µm C18 90 Å (Phenomenex), 250 mm × 4.6 mm, elution gradient 10% to 70% A in 40 min, 1 mL/min. HPLC system 2 for the analysis of the radioactive conjugates: Perkin-Elmer, consisting of a Turbo LC System with a quarternary pump (series 200 LC pump), a programmable absorbance detector model 785A (Perkin-Elmer Instruments, Germany), and a homemade γ-ray detector (well-type, NaI(Tl) crystal). Method 3: Jupiter 4 µm C18 90 Å (Phenomenex), 250 mm × 10 mm, elution 10% to 70% A in 20 min, 2 mL/min. Method 4: Jupiter 4 µm C18 90 Å (Phenomenex), 250 mm × 10 mm, elution gradient 10% to 70% A in 40 min, 3 mL/min. HPLC system 3 for the preparative

Hexadentate Bispidine Derivatives

sample purification: Knauer Wellchrom instrument, consisting of a pump K-1001, a UV detector U-2501, and a solvent organizer K-1500 for low-pressure gradient operation. Method 5: Zorbax 7 µm 300 SB-C18 Prep (Agilent), 250 mm × 21.2 mm, elution gradient 10% to 70% A in 30 min, 8 mL/min. Dimethyl-9-hydroxy-2,4-dipyridin-2-yl-3,7-bis(pyridin-2ylmethyl)-3,7-diazabicyclo[3.3.1]-nonane-1,5-dicarboxylate (2). 1 (500 mg, 0.844 mmol) was dissolved in 8.3 mL dioxane and mixed with 5.8 mL of water. Under ice cooling and stirring, 216 mg (5.71 mmol) sodium borohydride dissolved in 4.5 mL water was dropped into this solution. The reaction mixture was stirred 24 h at room temperature. The pH was set to 1 with dilute hydrochloric acid, the solvent was removed under reduced pressure, and the pH was set to 8 with a 1 M sodium hydroxide solution. The mixture was stirred for 1 h at room temperature and extracted five times with dichloromethane. Crystalline material suitable for X-ray analysis could be obtained from methanol. For further characterization, the crude product was purified by RP-HPLC (method 5) to yield 2 as a white solid after lyophilization. Yield: 276 mg (55%). Mp: 167 °C. Anal. Found (%): C, 64.84; H, 6.03; N, 13.36. Calcd for C33H34N6O5 · 1H2O (%): C, 64.69; H, 5.92; N, 13.72. ESI-MS (m/z): 595.4 (100%) [M + H]+. HPLC (method 1): tR ) 9.4 min. 9-Hydroxy-2,4-dipyridin-2-yl-3,7-bis(pyridin-2-ylmethyl)3,7-diazabicyclo-[3.3.1]nonane-1,5-dicarboxylic acid (3). To 200 mg (0.336 mmol) of 2, dissolved in 6 mL of anhydrous tetrahydrofurane, was added 565 mg (3.36 mmol) cesium hydroxide. Under ice cooling, 34 µL water was added, and after stirring 24 h at room temperature, a milky white emulsion was obtained. After setting the pH to 6 with dilute hydrochloric acid, the solvent was removed in vacuo. The crude product was dissolved in ethanol and purified by preparative RP-HPLC (method 5) to yield 3 as a white solid after lyophilization. Yield: 166 mg (87%). Mp: 174 °C. Anal. Found (%): C, 63.34; H, 5.83; N, 14.32. Calcd for C31H30N6O5 · 1H2O (%): C, 63.69; H, 5.52; N, 14.37. ESI-MS (m/z): 567.3 (100%) [M + H]+. HPLC (method 1): tR ) 8.1 min. Diethyl 4,4′-{[9-Hydroxy-2,4-dipyridin-2-yl-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo-[3.3.1]nonane 1,5-diyl]bis(carbonylimino)}dibutanoate (4). 50 mg (0.088 mmol) of 3 and 66.9 mg (0.176 mmol) HBTU were dissolved in 500 µL dry dimethylformamide. Subsequently, 76 µL (0.412 mmol) DIPEA and 20 mg (0.154 mmol) ethyl-4-aminobutyrate, dissolved in 1 mL dry dimethylformamide, were added under ice cooling, and the reaction mixture was stirred for 3 h at room temperature. The solvent was removed under reduced pressure, and the crude product was purified by RP-HPLC (method 5) to yield 4 as a white solid after lyophilization. Yield: 14 mg (17%). Anal. Found (%): C, 53.34; H, 5.43; N, 10.62. Calcd for C43H52N8O7 · 2H2O · (TFA)2 (%): C, 53.41; H, 5.53; N, 10.60. ESI-MS (m/z): 793.6 (100%) [M + H]+. HPLC (method 2): tR ) 18.5 min. 4,4′-{[9-Hydroxy-2,4-dipyridin-2-yl-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo-[3.3.1]nonane-1,5-diyl]bis(carbonylimino)}dibutanic Acid (5). To 20 mg (0.019 mmol) of 4, dissolved in 500 µL of anhydrous tetrahydrofurane, was added 32 mg (0.19 mmol) cesium hydroxide. Under ice cooling 5 µL water was added, and after stirring 24 h at room temperature, a milky white emulsion was obtained. After setting the pH to 6 with dilute hydrochloric acid, the solvent was removed in vacuo. The crude product was dissolved in ethanol and purified by preparative RP-HPLC (method 5) to yield 5 as a white solid after lyophilization. Yield: 17 mg (90%). Anal. Found (%): C, 51.64; H, 5.00; N, 11.18. Calcd for C39H44N8O7 · 2H2O · (TFA)2 (%): C, 51.60; H, 5.04; N,

Bioconjugate Chem., Vol. 20, No. 2, 2009 349

11.20. ESI-MS (m/z): 737.4 (100%) [M + H]+. HPLC (method 2): tR ) 13.4 min. N-{6-[5-(5-Carboxypentanoyl)-9-hydroxy-2,4-dipyridin-2yl-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo-[3.3.1]non-1yl]-6-oxohexanoyl}-βhomo-Glu-βAla-βAla-[Cha13,Nle14]BBN(714) (6). Five milligrams (6.78 µmol) of 5 and 2.6 mg (6.78 µmol) HATU were dissolved in 400 µL anhydrous dimethylformamide and stirred for 10 min at room temperature. Afterward, 7 equiv of DIPEA (4.1 mg, 0.032 mmol) and 5.5 mg(4.52µmol)βhomo-Glu-βAla-βAla-[Cha13,NIe14]BBN(7-14) in 300 µL anhydrous dimethylformamide were added under ice cooling. The mixture was stirred for 3 h at room temperature, and the solvent was then removed in vacuo. The residue was dissolved in acetonitrile/water (1:1) and purified by RP-HPLC (method 5) to yield 6 as a white solid after lyophilization. Yield: 2.5 mg (19%). ESI-MS (m/z): 1988.94 (100%) [M + Na]+. High-resolution ESI-TOF-MS (m/z): Found 983.5108 (45%); calcd 983.5103 for [M + 2H]2+. HPLC (method 2): tR ) 19.9 min. Cu-2(NO3)2 · H2O. To 100.5 mg (0.169 mmol) of 2, dissolved in 2 mL methanol, was added a solution of 31.5 mg (0.169 mmol) Cu(NO3)2 in 1 mL methanol. The mixture was stirred for 30 min (r.t.). The color changed from pale to dark blue. Slow diffusion of diethyl ether into this solution produced crystals that were suitable for X-ray crystallography. Yield: 29.7 mg (22%). Anal. Found (%): C, 49.68; H, 4.61; N, 14.28. Calcd for C33H34N8O11Cu · 1H2O (%): C, 49.53; H, 4.53; N, 14.00. ESIMS (m/z): 656.4 (100%) [M + H]+. HPLC (method 1): tR ) 10.2 min. Cu-3(NO3)2 · 2H2O. To 95.7 mg (0.169 mmol) of 3, dissolved in 2 mL methanol, was added a solution of 31.5 mg (0.169 mmol) Cu(NO3)2 in 1 mL methanol. The mixture was stirred for 30 min (r.t.), changing the color from pale to dark blue. Slow diffusion of diethyl ether into this solution produced crystals that were suitable for X-ray crystallography. Yield: 34.7 mg (26%). Anal. Found (%): C, 47.31; H, 4.51; N, 14.08. Calcd for C31H30N8O11Cu · 2H2O (%): C, 47.12; H, 4.34; N, 14.18. ESIMS (m/z): 629.4 (100%) [M - H]-. HPLC (method 1): tR ) 9.7 min. Cu-6(TFA)x. To 10 mg (0.0051 mmol) of 6 dissolved in 1 mL methanol was added a solution of 1 mg (0.0051 mmol) Cu(NO3)2 in 0.5 mL methanol. The mixture was stirred for 24 h, the solvent was then evaporated, and the resulting oily residue was purified RP-HPLC (method 3) to yield a pale blue solid after lyophilization. Yield: 6.4 mg (58%). ESI-MS (m/z): 1014.47 (100%) [M + H]2+. High-resolution ESI-TOF-MS (m/z):Found 1013.9698 (35%); calcd 1013.9673 for [M + Cu]2+. HPLC (method 3): tR ) 12.9 min. X-ray Data Collection, Processing, and Structural Determination of Cu-1 and Cu-2. The X-ray data were collected at room temperature (293 K) on a SMART-CCD diffractometer (SIEMENS), using graphite monochromatized Mo KR radiation (λ ) 0.710 73 Å). The structures were solved by direct methods with SHELXS-97 (G. M. Sheldrick, Universita¨t Go¨ttingen, 1997). A full matrix least-squares refinement on F2 was carried out using SHELXL-97. The hydrogen atoms were assigned with common isotropic displacement factors and included in the final refinement by the use of geometrical restrains. Further details of X-ray structure analyses are given in Table 1. Selected bond lengths and angles are listed in Table 2. CCDC 705478 (2), CCDC 705479 (Cu-2), and CCDC 705480 (Cu-3), contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/ cif, by e-mailing [email protected], or by con-


Juran et al.

Table 1. Comparison of Selected Bond Lengths [Å] and Angles [°] of Cu-1, Cu-2, and Cu-3 bond lengths [Å] Cu-N3 Cu-N7 Cu-N(py(C2)) Cu-N(py(C4)) Cu-N(py(N3)) Cu-N(py(N7)) ∑i6) 1 (Cu - ni)

Cu-1 (14)

Cu-2

Cu-3

2.093(3) 2.038(3) 2.262(3) 2.608(3) 2.012(3) 2.031(3) 13.045(3)

2.063(5) 2.028(5) 2.387(6) 2.538(5) 1.976(5) 2.001(5) 12.993(5)

2.064(5) 2.028(5) 2.333(5) 2.551(5) 1.986(5) 2.016(5) 12.978(5)

2.818(5) 4.719(5)

2.803(5) 4.714(5)

distances [Å] N3-N7 N(py(C2))-N(py(C4))

2.840(3) 4.696(4) angles [°]

N3-Cu-N7 N3-Cu-N(py(C2)) N3-Cu-N(py(C4)) N3-Cu-N(py(N3)) N7-Cu-N(py(N7)) N3-Cu-N(py(N7)) N7-Cu-N(py(N3))

86.87(12) 78.54(11) 72.79(13) 84.64(11) 82.96(12) 153.31(12) 171.34(13)

87.1(2) 75.7(2) 73.6(2) 86.0(2) 83.7(2) 157.0(2) 171.5(2)

86.5(2) 77.0(2) 74.23(2) 86.6(2) 84.6(2) 156.5(2) 171.2(2)

Table 2. Spectroscopic and Electrochemical Data of Copper(II) Complexes with Ligands 1-3 and Cyclam Cu-1

Cu-2

Cu-3

Cu-cyclam

UV/vis (dd transition)a 620 (110)b 620 (100) 614 (110) 510 (93)c -573 f E° (CuII/I)d (mV) -596 -690 -431 -502 f E° (CuII/I)e (mV) -415 -486 a λmax [nm] (ε [M-1 cm-1)]. b From ref 14. c From ref 21. d In acetonitrile, vs Ag/AgNO3; µ ) 0.1. e In water, vs Ag/AgNO3; µ ) 0.1. f From ref 15.

tacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033. Preparation of Radioactive Copper Complexes 64Cu-1, 64 Cu-2, 64Cu-3, and 64Cu-61. To 200 µL of ligand solution (0.1 mM of 1-3 in acetonitrile/0.1 M NH4OAc ) 1/1), an aqueous solution of 64CuCl2 (500 kBq, 100 µL, 0.1 M NH4OAc) was added. The labeling efficiency was investigated as a function of time and ligand concentration. Radiochemical purity was determined by HPLC (method 3) and radio-TLC. For radiolabeling of the bioconjugate 6, 0.5 mg ligand dissolved in 100 µL acetonitrile/water (1/1) 100 µL 64CuCl2 (5-20 MBq, 0.1 M NH4Oac, pH 6.5) was added. Incubation at 37 °C for 30 min yielded g99% radiochemical purity (HPLC, method 3). Ligand Exchange “Challenge” Studies. UV/vis spectroscopy was used to study the stability of the copper(II) complexes with ligands 1-3 in the presence of glutathione. To the copper(II)bispidine complexes (2 mM in water), 50 equiv glutathione (0.1 M) was added, and the electronic absorption spectra were recorded in the range from 400 to 800 nm as function of time (each 1 h for 24 h). To study the influence of cyclam as competing ligand, radiotracer experiments with 64Cu were used. 250 µL 64CuCl2 (500 kBq) in 0.1 M NH4OAc was added to 500 µg 1-3 dissolved in 100 µL acetonitrile/water (1/1). After 15 min, full complexation of the ligand was checked by TLC. Then, 40 mg cyclam (0.2 mmol; more than 1000-fold excess (1000-10 000-fold excess, depending on varying the specific activity of different Cu-64 production runs and time of Cu-64 use after EOB) dissolved in 1 mL acetonitrile/water (1/1) was added to the complex solution, buffered to pH 6 by addition of acetic acid, and stirred for 24 h. The degree of decomposition was determined by radio-TLC and radio-HPLC (method 3) using aliquots of the mixtures. 1 Charges and oxidation states are all omitted in this nomenclature; all complexes investigated here are CuII complexes; 64Cu-1, e.g., is a 2+ charged cation.

SOD “Challenge” Studies. 250 µL 64CuCl2 (5 MBq) in 0.1 M NH4OAc was added to 10 µg of the ligands 1-3 dissolved in 100 µL acetonitrile/water (1/1). The complete complexation of the ligand was checked by TLC after 15 min. After removal of the acetonitrile, more than a 1000-fold excess of SOD (5 mg, from bovine liver dissolved in 200 µL of 0.9 M NaCl solution) was added to the complex solution and stirred for 24 h. The degree of decomposition was determined by radio-TLC, using aliquots of the mixture. Determination of the Partition Coefficient log Do/w. Compounds 2, 3, 5, and 6 were dissolved in 50 µL water (c0 ) 1 mM) and added to a mixture of 390 µL HEPES/NaOH buffer (0.05 M, pH ) 7.2, 7.4, 7.8), 10 µL 64CuCl2 solution in 0.1 M NH4OAc and 50 µL 10-4 M Cu(NO3)2 in water. Due to the low water solubility of ligand 1, the corresponding Cu(II) complex was used. The distribution experiments in 1-octanol/ buffer systems were performed at 25 ( 1 °C in microcentrifuge tubes (2 cm3) by means of mechanical shaking for 30 min. The phase ratio V(1-octanol):V(aq) was 1:1 (0.5 cm3 each). All samples were centrifuged, the phases separated, and the copper concentration was determined in both phases radiometrically using γ-radiation [64Cu, NaI(Tl) scintillation counter automatic gamma counter 1480, Wizard 3”, PerkinElmer]. In Vitro Studies. To 500 µg of the ligands (1-3, 6), dissolved in 50 µL water/acetonitrile (1:1), 10 MBq of 64CuCl2 was added. After complexation, 250 µL phosphate buffer (0.1 M, pH ) 7.4) and 250 µL rat plasma were added, and the solution was incubated at 37 °C for 2 and 24 h, respectively. For precipitation of blood cells and proteins, cold ethanol (500 µL) was added and the supernatant was centrifuged two times. The ethanolic solution was dried under nitrogen, dissolved in 100 µL water/ acetonitrile (4/1 + 0.1% TFA), the residue filtered off, and the filtrate checked by HPLC (method 3). In ViVo Stability. The in vivo stability of 64Cu-6 was analyzed using rat arterial blood samples at various time points after intravenous injection of the radiotracer. For rat 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 intravenous tracer application. 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 radio-HPLC, Zorbax (Agilent) SB-C18, 300 Å, 5 µm, 250 × 9.4 mm; gradient elution using eluents C (acetonitrile containing 0.05% TFA) and D (water containing 0.04% TFA), 10% to 70% eluent C in 20 min, 3 mL/min, 28 °C. The chromatogram of 64Cu-6 (plasma, 0 min) was obtained by addition of 64Cu-6 to rat blood and immediately treating the mixture as described for the in vivo samples. Urine and kidney homogenate (10% in PBS) were treated analogously to the arterial blood samples. Animals, Feeding, Husbandry, and Biodistribution Studies in Rats. The animal research committee of the Landesdirektion 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 (body weight 227 ( 10 g). 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. The NMRI nu/nu mice were housed under similar conditions except temperature (27 ( 1 °C) in controlled-airflow cabinets. Animals for each time point were intravenously injected into a tail vein with approximately 8 µCi (0.3 MBq) 64Cu-3 or 64Cu-6 conjugate in 0.5 mL electrolyte



Scheme 3. Synthesis of 4 and 5 by Introduction of an Aminobutyric Acid Linkage into 3 to Facilitate the Attachment of Biomolecules into the Bispidine Scaffold (py ) pyridin-2-yl)a

a (a) ethyl-4-aminobutyrate, HBTU, DIPEA, DMF, 0 °C - r.t. 3 h, 17%, (b) CsOH, THF, 0 °C - r.t., 24 h, 90% after recrystallization from ethanol.

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 decaycorrected 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). General anesthesia of NMRI nu/nu mice was induced with inhalation of desflurane 12% (v/v) (Suprane, Baxter, Germany) in 40% oxygen/air (gas flow 1 L/min) and was maintained with desflurane 8% (v/v). In the PET experiments, approximately 0.5 mCi (18.5 MBq) of the 64Cu-6 conjugate in 0.5 mL was administered intravenously over 1 min into a tail vein. PET imaging of 64Cu-6 in mice was performed over 60 min with a microPET P4 scanner (Siemens CTI Molecular Imaging Inc., Knoxville). Data acquisition was performed in 3D list mode. For estimation of photon attenuation in the subjects, a transmission scan was carried out prior to the injection of 64Cu-6 using a 57Co point source. Emission data were collected continuously for 60 min after injection of 64Cu6. The list mode data were sorted into sinograms using a framing scheme of 12° s 10 s, 6° s 30 s, 5° s 300 s, and 5° s 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 β-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 64Cu standards from the injection solution measured in a µ-well counter (Isomed 2000, Dresden, Germany) crosscalibrated 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 Cu-2 > Cu-3, indicating increasing stability from the bispidine complex (Cu-1) to the bispidole (Cu-2) and the bispidole dicarboxylic acid

complex (Cu-3) (see above). It is worth mentioning that the reduction potentials of the copper(II) complexes with bispidine ligands are significantly lower than those of the cyclam complex, which forms one of the most stable copper complexes (log K1 )


Figure 4. Examples of radio chromatograms of original 64Cu-2 (A) and of rat plasma supernatant 60 min after application of 64Cu-2 (B).

27.2) (46, 47). Note that the correlation of Cu(II/I) redox potentials with Cu(II) complex stabilities relies on the assumption of nearly constant Cu(I) stabilities, and this has been shown to be an oversimplification (20). Radiochemistry. Preliminary experiments showed that the rate of copper(II) binding by bispidine ligands is very rapid at pH 6.5 and independent of the buffer system used (there is no difference among acetate, succinate, citrate, and 2-[N-morpholino]ethanesulfonic acid (MES) buffer, respectively). That is in contrast to the 64Cu-labeling of tetraaza macrocycles, where relatively slow complexation kinetics have been observed in acetate buffer solution (48). 64Cu-labeling of ligands 1-3 and 6 has been studied in detail in aqueous ammonium acetate solution. To 200 µL of 0.1 mM ligand dissolved in acetonitrile/NH4OAc (1/1), an aqueous solution of 64CuCl2 (500 kBq, 100 µL, 0.1 M NH4OAc) was added. The labeling yields were monitored by thin-layer chromatography using neutral alumina plates, which were developed in 0.1 M ammonium acetate/methanol (1/1). The Rf-values of free copper and 64Cubispidine complexes are well-separated. 64CuCl2 remained at the origin, and the 64Cu-bispidine complexes move further with Rfvalues of 64Cu-1, Rf ) 0.56; 64Cu-2, Rf ) 0.52; 64Cu-3, Rf ) 0.46. The bispidine ligands 1-3 were quantitatively labeled with 64Cu within 1 min at ambient temperature under these experimental conditions. Further experiments revealed that only 1 µg of ligand is necessary to obtain complete copper complexation (5 MBq 64Cu). 64 Cu-labeling of the bispidine bioconjugate 6 is slower than that of the unmodified ligands. Binding of copper(II) in an initial complexation step by peptide donor groups (imidazole) may lead to slower complex formation. Complexation of 64Cu by 6 is complete after approximately 1 h at room temperature and 30 min at 37 °C. Altogether, a very efficient and rapid 64Cu complexation of bispidine derivatives was found under mild conditions. Challenge experiments in the presence of cyclam and the enzyme superoxide dismutase (SOD) as competing ligands have been performed to obtain further information about the stability of the radio copper bispidine complexes. HPLC (method 3) and TLC studies using neutral alumina plates (see Experimental Section) revealed that the complexes 64Cu-1, 64Cu-2, and 64Cu-3 remained completely intact even in a 1000-fold excess of cyclam for at least 24 h. The stability of the radio copper complexes of these ligands was also investigated in the presence of SOD, a homodimeric enzyme which is abundant in liver, kidney, adrenal, and red blood cells. The competition assay using SOD can be used to predict the in vivo stability of radio copper

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complexes (49, 50). The challenge studies have been performed with more than 1000-fold excess of SOD compared to the 64Cu bispidine complexes. The degree of decomposition was checked via TLC (neutral alumina) that allows a clear distinction of the radio copper complexes formed (64Cu-SOD, Rf ) 0; 64Cu-1, Rf ) 0.56; 64Cu-2, Rf ) 0.52; 64Cu-3, Rf ) 0.46). Control experiments using RP-C18 plates (ethanol/MES-NaOH-buffer containing 0.001 M EDTA ) 5:1) showed that free copper does not occur in the solution (64Cu-SOD, Rf ) 0; 64Cu-EDTA, Rf ) 0.69). The absence of SOD-bound radioactivity indicates no transchelation of copper(II) and emphasizes a very high stability of the copper(II) bispidine complexes. This is also confirmed by rat plasma stability studies. The plasma stability of 64Cu complexes of ligands 1-3 and 6 was evaluated at 2 and 24 h. Radio-HPLC experiments showed that copper(II) is very tightly bound to the bispidine moiety. No demetalation of 64Cu was found in rat plasma up to 24 h. In the case of the dicarboxylate 3, the radio copper bispidine complex remains completely unchanged after rat plasma incubation. In contrast, for the radio copper complexes of the ester derivatives 1 and 2 two additional peaks appear in the chromatogram, resulting from the corresponding monoester and dicarboxylate caused by cleavage of the ester groups by esterases of the rat plasma (51). Furthermore, the in vitro stability studies reveal that peptidases of rat plasma attack the bombesin residue of the bioconjugate 6 (cf. in vivo stability studies). To facilitate the interpretation of biodistribution data, the partition coefficients of the radio copper complexes have been determined in a 1-octanol/buffer system (Table 3). The copper(II) bispidine complexes are rather hydrophilic. As expected, the introduction of an aminobutyric acid spacer (64Cu-5) leads to increased lipophilicity. The radio copper complexes of the bombesin-bispidine conjugate 64Cu-6 and the bombesin-bis(2pyridylmethyl)-triazacyclononane derivative (log Do/w ) -2.38, pH ) 7.4) possess almost the same hydrophilicity (23). Therefore, comparative biodistribution behavior, such as rapid blood clearance and excretion via the renal pathway, are expected for these 64Cu-labeled pyridine-containing ligands. In Vivo Studies. Due to the high affinity of the peptide bombesin to gastrin-releasing peptide receptors (GRPr), the design and synthesis of radiolabeled bombesin derivatives has recently attracted much attention (52-55). GRPs are overexpressed on a variety of human cancer cells, such as lung, breast, pancreatic, and prostate tumors. Therefore, the development of targeting vectors based on radiolabeled bombesin derivatives for imaging (PET, SPECT) and targeted radionuclide therapy is of considerable interest. Most investigations have been performed with 99mTc-labeled bombesin peptides, and to a lesser extent also with 18F, 68Ga, 125I, 111ln, 177Lu, 188Re, 86Y, and 90 Y (33, 52-65). Due to its favorable decay properties, the increasing availability, and the capability of producing highquality PET images, 64Cu is gaining importance in bombesin labeling (23, 66-74). In vivo studies performed so far have shown that the pharmacokinetics of the radiolabeled bombesin derivatives are influenced by the radio isotopes chosen, the chelate unit, the spacer elements, the specific modifications made to the peptide sequence and the species selected. Overall, predictions about the biodistribution of appropriate radiotracers are difficult. Therefore, we have investigated in some detail the biodistribution of various 64Cu-labeled bispidine derivatives and the metabolism of the bispidine-bombesin conjugate 6, and small-animal PET studies have also been performed. A summary of the biodistribution and elimination data for 64 CuCl2 (A), 64Cu-2 (B), 64Cu-3 (C), 64Cu-6 (D) in rats is shown in Figures 2 and 3. The numeric values of %ID (% of total injected dose) and SUV (standardized uptake value) are assembled in the Supporting Information.



Figure 5. Radio HPLC of 64Cu-6 metabolites in vitro at 1 min (A) and 60 min (B), and 60 min after application in arterial blood plasma (C), kidney homogenates (D), and urine (E).

Figure 6. Coronal sections of a small animal PET study of PC-3 tumor-bearing mouse after single intravenous application of 64Cu-6 at 0.5 (A), 5 (B), and 45 min (C, high intensity; D, low intensity).

The application of 64CuCl2 resulted mostly in the accumulation of radioactive species in liver and intestine, but also in significant radioactivity retention in the kidneys. The bispidine complexes 64 Cu-2 and 64Cu-3 were mainly eliminated into the urine as the original compounds. The metabolic analysis of 64Cu-2 and 64Cu-3 emphasizes the high stability of the complexes in vivo (cf., Figure 4). The initial accumulation of these compounds was similar to 64 CuCl2 in the liver, but surprisingly, the more hydrophilic acid derivative 64Cu-3 was also hepatobiliary eliminated (the sum of liver and intestine activity was 26.56 ( 1.79% ID, which means more than 50% activity eliminated or on excretion pathway). The

highest uptake and the resulting high activity concentration (SUV) was observed in the kidney for all compounds studied. However, the fastest elimination of radioactive species was observed for the bombesin conjugate 64Cu-6. In rats, low uptake of 64Cu-6 in the pancreas, an accessible GRPr-expressing tissue, was observed. This behavior could have been caused by different effects, such as fast metabolism of the peptide residue and rapid excretion of the radiotracer. Bombesin conjugates bearing 99mTc(III) ‘4 + 1’ complexes showed similar lack of pancreas accumulation (64). The metabolic stability of 64Cu-2 and 64Cu-6 was determined in arterial blood samples taken at 1, 3, 5, 10, 20, 30, and 60


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Figure 9. Time course of tumor to muscle relation of 64Cu-6 in PC-3 tumor bearing nude mice (mean ( SD two animals) of control (circles) and blocked (square, 1 mg natCu-6/kg body weight, simultaneously administered with 64Cu-6).

Figure 7. Coronal sections of a small animal PET study of two PC-3 tumor-bearing mice (A,B) after single intravenous application of 64Cu-6 at 5 and 45 min. The arrow shows the tumor.

Figure 8. Biokinetics of 64Cu-6 in PC-3 tumor bearing nude mice (mean ( SD of two animals). Time-activity curves were derived from ROIs over the aorta, tumor, and muscle, expressed as SUVmean.

min after a single intravenous application of the radiotracer into a rat. The blood samples were centrifuged to obtain the plasma, which was treated with acetonitrile to precipitate the proteins. The supernatant is referred to as the plasma extract. After 64Cu-2 was applied, the mean relative radioactivity concentration in the plasma was 90 ( 2% of the overall arterial blood activity; the plasma extract samples contained 84 ( 3% of the plasma activity. Furthermore, the plasma extract was analyzed by radio RP-HPLC for radio metabolites. Figure 5 shows the radio chromatograms of the original compound 64Cu-2 and the plasma analysis after 60 min. The main peak of in vivo samples, assigned to the original 64 Cu-2 complex, accounts for 98% and after 60 min for 97% of the activity (see Figure 4). The radiochromatograms of other plasma and also of urine samples are very similar. The mean relative activity of 64Cu-6 in plasma was 88 ( 5% of arterial blood activity; the activity of the plasma extract samples contained 66 ( 12% of the plasma activity. The plasma extract was analyzed by RP-HPLC for radiometabolites (Figure 5).

The relative amount of the original compound 64Cu-6 (tR ) 13.9 min) decreased from 96% at the time of tracer application (1 min 96%, 3 min 89%, 5 min 80%, 20 min 44%, 30 min 38%) to 3% of the total plasma extract activity after 60 min. 64 Cu-6 was converted into three more hydrophilic radiolabeled species. The peak at 3.8 min is not due to free 64Cu2+ ions, since these are eluted at 3.4 min. The mixture of residual radioactive species in the analyzed tissues and urine was very complex. Overall, the detected radiometabolites were more hydrophilic than the native compound. The original 64Cu-6 complex could not be detected in kidney extracts or urine. Extensive metabolism of 64Cu-6 has occurred in both species studied (rats and tumor-mice). This is in contrast to the in vitro stability. In vitro incubation of 64Cu-6 in rat blood or blood plasma yielded more than 85% of the original compound after 60 min in the nonprecipitated plasma fraction. However, the decomposition of up to 15% in vitro, which is due to partial hydrolysis, seems to be a sign of a specific effect of the bispidine complex units. A similar bombesin derivative, labeled with 64Cu using 2-[4,7-bis(2pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid as bifunctional chelate, showed higher metabolic in vitro stability under analogous conditions (23). Preliminary PET studies in PC-3 tumor-bearing mice have also been performed. The bombesin derivative 64Cu-6 was accumulated in the GRPr-expressing tissue (see Figures 6 and 7) to allow the visualization of the tumor in the right axilla. The highest radiotracer concentration in the unblocked tumors was reached at 12.5 min (2.25 ( 0.13 SUV). However, the activity was very rapidly washed out from the tumor (Figure 8). Therefore, a high intensity is required to distinguish the tumor after 45 min, and this leads to a high background level (Figure 6C). Nonetheless, high-contrast PET images of the tumor could be produced. The tumor to muscle ratio has been determined as function of time (Figure 9) and shows an increasing value for the unblocked (control) mice (1.78 ( 0.14 at 1 h) in contrast to the blocked animals (0.84 ( 0.01 at 1 h, 1 mg natCu-6/kg body weight). This finding points to a receptor-mediated tracer uptake into tumor tissue.

CONCLUSION Five new hexadentate pyridine-containing bispidine derivatives 2-6 have been synthesized. X-ray diffraction studies of the copper(II) complexes of 2 and 3 confirmed a distorted octahedral geometry with an efficiently encapsulated and shielded Cu2+ center. UV/vis spectra indicate that these cop-



Scheme 4. Coupling of 5 to the Bombesin Derivative Leads to Bioconjugate 6a

a

(a) βhomo-Glu-βAla-βAla-[Cha13, Nle14]BBN(7-14), HATU, DIPEA, DMF, 0 °C - r.t., 3 h, 19%.

Chart 1

Supporting Information Available: NMR data of compounds 2, 3, 4, and 5. Tables of crystal data, structure refinement, bond lengths and angles for compounds 2, Cu-2, and Cu-3, and atomic coordinates and equivalent isotropic displacement parameters for 2 and Cu-2 are presented. Figures S1 and S2 show the molecular structures of 2, Cu-2, and Cu-3. Biodistribution data of 64CuCl2, 64Cu-2, 64Cu-3, and 64Cu-6 in rats are presented as %ID and SUV. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED per(II)-bispidine complexes are stable in the presence of a high excess of glutathion and cyclam as competing ligands. Cyclic voltammetry with negative CuII/CuI redox potentials is in agreement with a high stability of the copper(II) complexes. The hexadentate bispidine ligands investigated here rapidly form stable radiocopper complexes under mild conditions in almost quantitative yield. Challenge experiments in the presence of superoxide dismutase and in vitro studies in rat plasma gave no evidence of transchelation or demetalation. The bispidine copper complexes 64Cu-2 and 64Cu-3 show in all experiments high stability in vitro and in vivo. The pendant carboxylate groups of the bispidine backbone offer good accessibility toward coupling reactions. With an aminobutyric acid spacer, the bombesin analogue as a vector molecule was conjugated to the bifunctional bispidine ligand 6. Radiolabeling of bioconjugate 6 with 64Cu was achieved at 37 °C for 30 min, yielding >99% radiochemical purity after HPLC. Comparative biodistribution and metabolic analysis of 64Cu-6 indicated that there was only marginal, if any, in vivo copper demetalation. On the other side, the stabilized bombesin derivative βhomo-Glu-βAla-βAla[Cha13,Nle14]BBN(7-14) is readily metabolized. The accumulation of the radiolabeled peptide conjugate 64Cu-6 in the GRPrexpressing pancreas is very low. However, PET studies on tumor-bearing PC-3 mice clearly revealed an accumulation of 64 Cu-6 in the GRPr-expressing tissue. The blockage study showed that natCu-6 was able to reduce the uptake in the receptor-rich tissue. Bifunctional hexadentate bispidine ligands represent a versatile platform for the development of new copper radiopharmaceuticals. The two pendant carboxylate groups of the bispidines discussed here hold promising potential to tune the charge of the radiocopper complexes and consequently to influence the biodistribution and pharmacokinetic properties. Moreover, the new bispidine ligands may be readily modified with appropriate vector molecules.

ACKNOWLEDGMENT The authors would like to thank Romy Schubert, Karin Landrock, Regina Herrlich, and Andrea Suhr for excellent technical assistance. Marion Kerscher and Madlen Matterna are acknowledged for the CV measurements, and Andreas Springer (FU Berlin) for high-resolution ESI-TOF-MS measurements.

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