Steps toward High Specific Activity Labeling of Biomolecules for

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Bioconjugate Chem. 2002, 13, 750−756

Steps toward High Specific Activity Labeling of Biomolecules for Therapeutic Application: Preparation of Precursor [188Re(H2O)3(CO)3]+ and Synthesis of Tailor-Made Bifunctional Ligand Systems Roger Schibli,*,† Rolf Schwarzbach,† Roger Alberto,§ Kirstin Ortner,§ Helmut Schmalle,§ Ce´cile Dumas,† Andre´ Egli,† and P. August Schubiger†,‡ Center for Radiopharmaceutical Science, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland, Department of Applied Biosciences, Swiss Federal Institute of Technology, CH-8057 Zurich, Switzerland, and Department of Inorganic Chemistry, University of Zurich, CH-8057 Zurich, Switzerland. Received October 18, 2001; Revised Manuscript Received February 25, 2002

Two kit preparations of the organometallic precursor [188Re(H2O)3(CO)3]+ in aqueous media are presented. Method A uses gaseous carbon monoxide and amine borane (BH3‚NH3) as the reducing agent. In method B CO(g) is replaced by K2[H3BCO2] that releases carbon monoxide during hydrolysis. Both procedures afford the desired precursor in yields >85% after 10 min at 60 °C. HPLC and TLC analyses revealed 7 ( 3% of unreacted 188ReO4- and 95% with [188Re(H2O)3(CO)3]+ under mild reaction conditions (PBS buffer, 60 °C, 60 min) at ligand concentrations between 5 × 10-4 M and 5 × 10-5 M. Thus, specific activities of 22-220 GBq per µmol of ligand could be achieved. Incubation of the corresponding Re-188 complexes in human serum at 37 °C revealed stabilities between 80 ( 4% and 45 ( 10% at 24 h, respectively, and 63 ( 3% and 34 ( 3% at 48 h postincubation in human serum depending on the chelating system. Decomposition product was mainly 188 ReO4-. The routine kit-preparation of the precursor [188Re(H2O)3(CO)3]+ in combination with tailormade ligand systems enables the organometallic labeling of biomolecules with unprecedented high specific activities.

INTRODUCTION

In the past years, the bioinorganic chemistry of rhenium has been developed toward novel applications in therapeutic nuclear medicine (1, 2). Interests in this field origin from the fact, that the two particle emitting radioisotopes Re-186 and Re-188 have excellent physical decay properties for therapeutic applications (Re-186: t1/2 ) 89 h, β- 1.07 MeV, γ 137 keV; Re-188: t1/2 ) 18 h, β2.12 MeV, γ 155 keV). Furthermore, due to the generator technique, Re-188 is nowadays readily available carrierfree and at any time (3). In analogy to Tc-99m, most of the currently applied Re-186/188 labeling techniques use precursors or complexes in higher oxidation states (4, 5). However, it is a fact that rhenium needs harsher conditions to be reduced from its original oxidation state +VII to lower oxidation states typically +V/+III. It is also frequently observed, that these rhenium complexes have * To whom correspondence should be addressed: Telephone: ++41-56-310-2837, Fax: ++41-56-310-2849, E-mail: [email protected]. † Paul Scherrer Institute. ‡ Swiss Federal Institute of Technology. § University of Zurich.

a higher tendency reoxidize, than their technetium analogues. Therefore, kinetically more inert complexes containing technetium and rhenium in the low oxidation state +I have received more attention, recently (6-10). Soft metal centers show an increased kinetic inertness and a low affinity for hard nitrogen and oxygen donor groups, readily present in the blood serum. This characteristic protects the complexes in vivo against ligand dissociation and ligand exchange. Our group has developed a fully aqueous-based kit preparation of the organometallic technetium precursor [99mTc(H2O)3(CO)3]+ under mild reaction conditions in the presence of gaseous carbon monoxide and sodium borohydrate (11). The formulation could be optimized in terms of kit-stability and commercial production due to substitution of both gaseous CO and NaBH4 by K2[H3BCO2] (12). However, till today the corresponding Re-186/188 precursor [*Re(H2O)3(CO)3]+ 1 was obtained only in low yields via the above-mentioned methods. In the present work we describe two simple and fully aqueous-based synthetic routes to obtain the precursor [188Re(H2O)3(CO)3]+ in high yields and with high specific activities. To translate the excellent specific activities on the labeling of biomolecules, tridentate bifunctional

10.1021/bc015568r CCC: $22.00 © 2002 American Chemical Society Published on Web 05/11/2002

Radiolabeled Biomolecules for Therapeutic Application

ligand systems tailor-made for the precursors [188Re(H2O)3(CO)3]+ have been synthesized. The first ligand system consists of bis[imidazol-2yl]methylamine chelate originally developed for copper(II) (13). To our knowledge this chelate system has never been tested and reacted with a soft, organometallic precursor. The second type of ligands consists of an iminodiacetic acid (IDA) moiety, which is well-known to form complexes with almost any metal of the fist and second transition row. Both chelating systems proved to be good candidates for facile bifunctionalization with spacers of different chain length bearing a terminal amino or carboxylic acid group for amidic linkage to carboxylic acid or primary amino group of biomolecules. On the other hand, they offer three potential coordination sites, forming multiple stable chelating rings with the rhenium-tricarbonyl center. The X-ray structures of two model complexes exemplify the coordinative features of these new ligand systems. In vitro plasma stabilities of the corresponding Re-188 complexes will be discussed. MATERIALS AND METHODS

Solvents for syntheses were purchased from Aldrich Chemical Co. or Fluka, Buchs, Switzerland and were dried according to standard methods. Glycine 2, 5-aminopentanoic acid 3, and 10-aminoundecanoic acid 4 were purchased from Fluka. The precursor (NEt4)2[ReBr3(CO)3] (14), N-tert-butyloxycarbonyldiaminopropane 5, and N-tert-butyloxycarbonyldiaminohexane 6 (15), bis(imidazol-2-yl)nitromethane 7 (16), bis(imidazol-2-yl)carboxymethylaminomethane 8a (13), and K2[H3BCO2] (12) were synthesized according to the literature. Na[188ReO4] was eluted from a 188W/188Re generator (Oak Ridge National Laboratories, Oak Ridge, TN) using 0.9% saline. The elution volume (approximately 30 mL) was reduced to a total of 5 mL (max. 14 GBq/mL) using the method of Blower (17). HPLC analyses were performed on a Merck-Hitachi L-7000-system equipped with an L-7400 tunable absorption detector and a Berthold LB 506 B radiometric detector. HPLC solvents consisted of aqueous 0.05 M TEAP (triethylammonium phosphate) buffer, pH ) 2.25 (solvent A) and methanol (solvent B). For the radiochemical analyses, a Macherey-Nagel C-18 reversed phase column (10 µm, 150 × 4.1 mm) was used. The HPLC system started with 100% of A from 0 to 3 min. The eluent switched at 3 min to 75% A and 25% B and at 9 min to 66% A and 34% B followed by a linear gradient 66% A/34% B to 100% B from 9 to 20 min. The gradient remained at 100% B for 2 min before switching back to 100% A. The flow rate was 1 mL/min. The thinlayer chromatography (TLC) system was performed using glass-backed silica gel plates (Merck 60F254, mobile phase of 99% methanol 1% concentrated HCl) and paper (Whatman No.1, mobile phase 99.5% methanol, 0.5% 6 M aqueous HCl). The plates were scanned with a Burkard RAYTEST RITA-3200 radioanalyzer. Radioactive samples were counted in a Camberra Packard COBRA II auto gamma well counter. Reactions with activities >500 MBq/mL have been performed in specially equipped lead-boxes with manipulators. Nuclear magnetic resonance spectra were recorded on a 300 MHz Varian Gemini 2000 spectrometer. The 1H and 13C chemical shifts are reported relative to residual solvent protons as a reference. IR spectra were recorded on a Perkin-Elmer FT-IR 16PC using KBr pellets. Elementary analyses were performed at the department of inorganic chemistry of the university of Zurich. Preparation of Precursor [188Re(H2O)3(CO)3]+, 1. Method A. A 5 mg amount of BH3‚NH3 was placed in

Bioconjugate Chem., Vol. 13, No. 4, 2002 751

10 mL glass vial. The vial was sealed with an aluminum capped rubber stopper and flushed with CO for 20 min. The generator eluate (1 mL, 3-14 GBq/mL) was mixed with 6 µL of concentrated H3PO4 (98%) prior to the injection in the reaction vial. The vial was incubated at 60 °C for 15 min. Pressure from the evolving H2 gas was balanced with a 20 mL syringe. The reaction was cooled on an ice bath. The final pH of the reaction solution was neutral. Yield: 85 ( 5% determined by means of HPLC and TLC. Method B. A 5 mg amount of BH3‚NH3 and 3 mg of K2[H3BCO2] were placed in 10 mL glass vial and flushed with nitrogen. The generator eluate (1 mL, 3-14 GBq/mL) was mixed with 6 µL of concentrated H3PO4 (98%) prior to the injection in the reaction vial. The vial was incubated at 60 °C for 15 min. Pressure from the evolving H2 gas was balanced with a 20 mL syringe. The reaction was cooled on an ice bath. The final pH of the reaction solution was neutral. Yield: 80 ( 5% determined by means of HPLC and TLC. Radiolabeling. The radioactive complexes were prepared according to the following general procedure: 450 µL of a solution of [188Re(H2O)3(CO)3]+ and 50 µL of a 10-3 M, and 10-4 M, respectively, 950 µL of a solution of [188Re(H2O)3(CO)3]+ and 50 µL of a 10-2 M, 10-3 M, and 10-4 M solution of the corresponding ligand in PBS buffer (0.1 M NaCl/0.05 M sodium phosphate buffered, pH ) 6.57.5) were placed in a 10-mL glass vial under nitrogen. The vial was sealed and the reaction heated to 60 °C for 60 min and cooled on an ice bath. In Vitro Plasma Stability. The solutions of the 188Re complexes were adjusted with physiological saline to a concentration of 370 MBq/mL. Aliquots of 100 µL of these solutions were added to 400 µL human plasma and incubated at 37 °C. Aliquots were taken and analyzed after 1 h, 4, 24, and 48 h. Preparation of Compound 8c and 8d. The ligands 8c/d were synthesized according to the procedure described for 8a (13) starting from the corresponding aminoalkyl carboxylic acid (n ) 4, 10). Yields: 50-70%. Analytical data for ligand 8c (n ) 4): Calculated for C12H17N5O2‚1.6 HCl: C 44.81; H 5.83; N 21.77. Found C 44.91; H 5.95; N 22.35. 1H NMR (D2O): δ 7.41 (s, 4 H), 6.68 (s, 1 H), 3.51 (t, 2 H), 2.33 (t, 2 H) 1.82 (m, 2 H), 1.69 (m, 2 H). 13C NMR (D2O): δ 177.9, 143.3, 122.7, 53.7, 42.0, 34.2, 25.6, 23.2. MS (ES): m/z (%) 264 (60) [M + 1]. Analytical data for ligand 8d (n ) 10): Calculated for C18H29N5O2‚1.7 HCl: C 52.80; H 7.56; N 17.10. Found: C 52.63; H 7.03; N 17.27. 1H NMR (D2O): δ 7.50 (s, 4 H), 6.30 (s, 1 H), 2.82 (t, 2 H), 2.23 (t, 2 H), 1.6-1.4 (m, 4 H), 1.2-1.1 (broad m, 12 H). 13C NMR (D2O): δ 179.1, 135.9, 122.9, 66.8, 47.8, 35.6, 34.0, 28.5, 25.7, 24.4, 23.3. MS (ES): m/z (%) 348 (100) [M + 1]. Preparation of Compounds 9a and 9b. The compounds 9a/b were synthesized according to the following, general procedure starting from the corresponding N-tertbutyloxycarbonyldiamines (n ) 3, 5, and n ) 6, 6). Bis[imidazol-2-yl]nitromethane (500 mg, 2.2 mmol) was dissolved in 2.6 mL of 2 M NaOH. One equivalent of the corresponding mono Boc-protected diaminoalkane was added under vigorous stirring. The suspension was heated at 80 °C for 20 min, in which a clear orange solution was formed. After cooling on an ice bath, the almost colorless product precipitated. The pure product was filtered and dried under vacuum. Yields: 50-70%. Analytical data for compound 9a (n ) 3): Calculated for C15H24N6O2: C 56.23; H 7.55; N 26.23: Found: C 55.96; H 6.95; N 26.71. 1H NMR (MeOH-d4): δ 6.91 (s, 4 H), 4.99 (s, 1 H), 3.06 (t, 2 H), 2.46 (t, 2 H), 1.56 (q, 2 H), 1.33 (s, 9 H). 13C NMR (MeOH-d4): δ 162.3, 143.4, 121.5,

752 Bioconjugate Chem., Vol. 13, No. 4, 2002

70.6, 51.6, 40.5, 33.8, 25.6, 24.2, 23.7. MS (ES): m/z (%) 321 (100) [M + 1]. Analytical data for compound 9b (n ) 6): Calculated for C18H30N6O2: C 59.64; H 8.34; N 23.19: Found: C 59.08; H 7.24; N 22.91. 1H NMR (MeOH-d4): δ 7.12 (s, 4 H), 5.01 (s, 1 H), 3.33 (t, 2 H), 2.53 (t, 2 H), 1.63 (m, 4 H), 1.40 (s, 4 H), 1.34 (s, 9 H). 13C NMR (MeOH-d ): δ 160.2, 144.4, 120.8, 66.2, 50.4, 4 42.5, 34.2, 28.6, 25.5, 25.1 23.4, 22.3, 20.1. MS (ES): m/z (%) 363 (100) [M + 1]. Preparation of Compounds 10a and 10b. Compounds 10a/b were deprotected in 3 M HCl at 50 °C for 20 min. After removal of the solvent, the products could be obtained almost quantitatively in the hydrochloride form as pale rose powder. Yields >95%. Analytical data for ligand 10a (n ) 3): Calculated for C10H16N6‚4.5 HCl: C, 31.0; H, 6.1; N, 21.7. Found: C, 31.1; H, 5.95; N, 21.5. 1H NMR (MeOH-d ): δ 7.58 (s, 4 H), 5.94 (s, 1 H), 3.16 4 (t, 2 H, 9 Hz), 2.81 (t, 2 H, 6 Hz), 1.95 (m, 2 H). 13C NMR (MeOH-d4): δ 139.6, 117.8, 57.7, 42.5, 39.4, 25.6. MS (ES): m/z (%) 221 (100) [M + 1]. Analytical data for ligand 10b (n ) 6): Calculated for C13H22N6‚4 HCl: C, 38.3; H, 6.4; N, 20.6. Found: C, 37.9; H, 6.1; N, 20.2. 1H NMR (MeOH-d4): δ 7.51 (s, 4 H), 6.04 (s, 1 H), 2.96 (t, 2 H), 2.73 (t, 2 H), 1.62 (m, 2 H), 1.58 (m, 2 H), 1.33 (m, 4 H). 13C NMR (MeOH-d4): δ 140.4, 117.7, 56.7, 42.5, 34.2, 28.6, 25.7,23.6, 20.4. MS (ES): m/z (%) 263 (80) [M + 1]. Preparation Compounds 11a and 11b. The compounds 11a/b were synthesized according to the following, general procedure using the corresponding amino alkyl carboxylic acid (n ) 4, 3 and n ) 10, 4). A 1 g amount of 5-aminopentanoic acid 3 or 11-aminoundecanoic acid 4, respectively, was dissolved in 100 mL of methanol together with 2.5 equiv of triethylamine. Methyl bromoacetate (2.1 equiv) was added over a period of 2 h at room temperature. The reaction was refluxed overnight. After removal of the solvent, the oily residue was purified by column chromatography (CH2Cl2/ethyl acetate: 4:1). The esters were saponified with 2 M NaOH under reflux for 60 min. The solution was neutralized with concentrated HCl and the solvent evaporated under reduced pressure. The residue was extracted twice with 5 mL of methanol to separate NaCl. After evaporation of the methanol the products were obtained as white solids. Yield: 40-60%. Analytical data for compound 11a (n ) 4): Calculated for C9H15NO6: C 46.35; H 6.48; N 6.01. Found: C 45.85; H 5.53; N 5.92. 1H NMR (MeOHd4): δ 3.38 (t, 2 H), 3.15 (s, 4 H), 3.04 (t, 2 H), 2.88 (q, 4 H). 13C NMR (MeOH-d4): δ 179.9, 171.5, 66.8, 56.2, 50.3, 33.1, 28.0. MS (ES): m/z (%) 234 (100) [M + 1]. Analytical data for compound 11b (n ) 10): Calculated for C15H27NO6: C 56.77; H 8.57; N 4.41. Found: C 56.45; H 6.97; N 4.21.1H NMR (D2O): δ 3.41 (t, 2 H), 3.21 (s, 4 H), 2.62 (t, 2 H), 1.77 (m, 2 H), 1.6-1.1 (m, 14 H). 13C NMR (D2O): δ 177.0, 171.0, 56.0, 51.1, 35.3, 33.2, 33.0, 28.6, 28.3, 26.8, 25.0, 23.3. MS (ES): m/z (%) 318 (100) [M + 1]. Preparation of Compounds 12a/b and 13a/b. The compounds 13a/b were synthesized according to the following, general procedure using the corresponding N-tert-butyloxycarbonyldiamines (n ) 3, 5 and n ) 6, 6). The N-tert-butyloxycarbonyldiamines were dissolved in 50 mL of methanol together with 2 equiv of triethylamine. Methyl bromoacetate (2.1 equiv) was added over a period of 2 h at room temperature. The reaction was refluxed overnight. After removal of the solvent the oily residue was diluted with water and extracted three times with ethyl acetate. The organic layers were combined and dried over Na2SO4. After filtration and evaporation of the solvent, the crude products 12a/b were checked by means of NMR and processed without purification. The crude

Schibli et al.

products were dissolved in 3 M HCl and heated at 50 °C for 30 min. After removal of the solvent the residues were dissolved in 2 M NaOH and refluxed for 60 min. The solution was neutralized with concentrated HCl and the solvent evaporated under reduced pressure. The residue was extracted twice with 5 mL of methanol to separate NaCl. After evaporation of the methanol, the products were obtained as white solids. Yield: 40-60%. Analytical data for ligand 13a (n ) 3): Calculated for C7H14N2O4‚ 0.3NaCl: C 40.47; H 6.79; N 13.49. Found: C 40.64; H 7.21; N 13.91. 1H NMR (D2O): δ 3.53 (s, 4 H), 2.83 (t, 2 H), 2.74 (t, 2 H), 1.87 (q, 2 H). 13C NMR (D2O): δ 176.1, 94.3, 64.1, 60.5, 51.7 40.0, 33.8. MS (ES): m/z (%) 191 (100) [M + 1]. Analytical data for ligand 13b (n ) 6): Calculated for C10H20N2O4‚0.2NaCl: C 49.23; H 8.26; N 11.48. Found: C 49.11; H 7.38; N 11.96. 1H NMR (D2O): δ 3.53 (s, 4 H), 2.83 (t, 2 H), 2.74 (t, 2 H), 1.87 (q, 2 H), 1.4-1.2 (m, 6 H). 13C NMR (D2O): δ 179.0, 95.1, 60.2, 58.1, 53.9, 42.3, 34.4, 29.6. MS (ES): m/z (%) 233 (100) [M + 1]. Preparation of Compound 8b. Compound 8a (200 mg, 0.9 mmol) was dissolved in 10 mL of methanol. The solution was cooled to 0 °C on an ice bath before adding 60 µL (0.9 mmol) of SOCl2. To the reaction solution was added 500 mg of molecular sieve, and the reaction was stirred overnight at room temperature. After filtration, the solvent was removed under reduced pressure to obtain the product. Yield 190 mg (90%). Analytical data: Calculated for C10H13N5O2: C 51.06; H 5.57; N 29.77. Found: C 51.12; H 4.98; N 29.24. 1H NMR (D2O): δ 7.12 (s, 4 H), 5.30 (s, 1 H), 3.67 (s, 3 H), 3.51 (s, 2 H). 13C NMR (D2O): 174.3, 138.1, 135.8 116.4, 56.7, 42.5. MS (ES): m/z (%) 236 (100) [M + 1]. Preparation [Re(8b)(CO)3]Br. (NEt4)2[ReBr3(CO)3] (100 mg, 0.12 mmol) was dissolved in 5 mL of methanol. One equivalent of 8a (30 mg) was added and the reaction stirred overnight at room temperature. The reaction was concentrated to 1 mL and layered with diethyl ether. The products crystallized completely after 3 days. Yield: 47 mg (65%). Analytical data: Calculated for C13H13N5O5ReBr: C 26.67; H 2.24; N 11.96. Found: C 26.12; H 2.98; N 11.24. 1H NMR (MeOH-d4): δ 7.40 (d, 1 H, 1.5 Hz), 7,33(d, 1 H, 1.5 Hz), 7.22 (d, 1 H, 1.5 Hz), 7.20 (d, 1 H, 1.5 Hz), 6.20 (s, 1 H), 3.78 (s, 3H), 3.61 (s, 2 H). IR (KBr, cm-1): 3420 (w), 2028 (vs), 1922 (vs), 1904 (vs), 1758 (m), 1290 (w), 1138 (m), 858 (w). Preparation of [Re(13aH+)(CO)3]. (NEt4)2[ReBr3(CO)3] (82 mg, 0.11 mmol) was dissolved in 5 mL of H2O. Ligand 13a (25 mg, 0.13 mmol) was added and the reaction heated at 70 °C for 2 h. Immediately after cooling to room temperature, the product crystallized almost quantitatively from the reaction solution. Yield: 48 mg (95%). Analytical data: Calculated for C10H13N2O7Re‚ 0.9H2O: C 25.25; H 3.14; N 5.89. Found: C 26.73; H 3.31; N 5.64. 1H NMR (DMSO-d6): δ 3.76 (d, 2 H, 16 Hz), 3.40 (d, 2 H, 16 Hz), 3.30 (t, 2 H), 3.28 (m, 2 H), 2.80 (t, 2 H). IR (KBr, cm-1): 3446 (w), 2362 (w), 2018 (vs), 1902 (vs), 1870 (vs), 1654 (s), 1622 (s), 1394 (m), 548 (w). RESULTS AND DISCUSSION

Contrary to the almost quantitative reductive carbonylation of 99mTcO4- in the presence of BH4-, CO, and Na2CO3 in aqueous media, only traces of the desired Re188 precursor were detected under these reaction conditions. Failure of this approach can presumably be explained by several facts: (i) Rhenium has a lower redox potential than technetium. (ii) Rhenium +V and/or +III intermediates presumably formed during the reductive

Radiolabeled Biomolecules for Therapeutic Application Scheme 1

carbonylation are unstable at basic pH. (iii) Under neutral/acidic conditions rhenium(III/IV) intermediates are stable but BH4- hydrolyzes fast. Thus, BH4- is not available for reduction of the metal center. To circumvent these problems, the water-soluble aminoborane, BH3‚ NH3, was used instead of BH4-. Aminoborane is sufficiently stable under neutral/acidic conditions and is highly reducing. The organometallic precursor [188Re(H2O)3(CO)3]+ 1 was synthesized via two routes starting from 188ReO4in saline. Method A uses solid BH3‚NH3 as the reducing agent and gaseous carbon monoxide as the source of CO ligands. The perrhenate solution was acidified with concentrated phosphoric acid prior to injection in the reaction vial to maintain an acidic/neutral pH during the reaction. After 15 min at 60 °C, the reaction was completed (Scheme 1). The HPLC γ-trace revealed beside the peak of the desired product 1 (70 ( 10%; RT ) 5 min) and remaining 188ReO4- (7 ( 3%; RT ) 10 min) a third peak of a byproduct with a retention time of 10.5 min (10 ( 5%). This byproduct of unknown composition can be converted into the precursor 1 by acidifying the carbonyl solution to pH < 2. TLC analysis of the carbonyl solution revealed only the peak of precursor 1 (85 ( 5%; Rf ) 0.4) and 188ReO4- (10 ( 2%; Rf ) 0.7). Less than 5% colloidal 188ReO2 was detected at the origin. In method B, K2[H3BCO2] was used as a solid source of CO and BH3‚NH3 as the reducing agent. The perrhenate solution was also acidified with concentrated phosphoric acid previous to injection in the reaction vial. After 15 min at 60 °C, the yields and product distribution were similar to those in method A (Scheme 1). The amount of BH3‚NH3/K2[H3BCO2] and acid (concentrated H3PO4) was carefully balanced, to avoid fast hydrolysis of the boranes and to maintain a sufficient low pH to stabilize reduced rhenium intermediates. Using exclusively K2[H3BCO2] instead of BH3‚NH3 and CO(g) did not yield the desired precursor even if applied in large excess (10-50 mg per reaction vial). Furthermore, it was observed that, when reaction volume was increased (>1 mL), the yields of 1 dropped significantly. Since a concerted mechanism, where the reduction of the metal center and the CO addition take place almost simultaneously, is proposed for the carbonylation reaction (12), the availability of reducing agent and CO are limiting factors. Thus, a fast saturation of the aqueous solution with carbon monoxide can only be guaranteed if the volume of the solution is relatively small. Yet, solutions of up to 14 GBq/mL Re188 could be successfully carbonylated with both methods. The free precursor 1 is stable (>90%) for approximately 3 h at pH ) 7-5. After this time, decomposition of the precursor and reformation of 188ReO4- was observed in the absence of effectively coordinating and stabilizing ligands. Addition of radical scavenging coligand such as gentisic acid or ascorbic acid did not significantly improve the half-life of the precursor.

Bioconjugate Chem., Vol. 13, No. 4, 2002 753 Scheme 2a

a Reagents and conditions: (a) 2 M NaOH, 80 °C; (b) MeOH, SOCl2; (c) 3 M HCl, 50 °C.

Scheme 3a

a Reagents and conditions: (a) MeOH, NEt , reflux, 15 h; 2 3 M NaOH; (b) 3 M HCl, 50 °C.

To translate the excellent yields and specific activities of precursor 1 on the radiolabeling of biomolecules such as peptides and proteins, novel, bifunctional ligand systems have been developed. Ligands 8 and 10 could be produced in a straightforward synthetic approach. Bifunctionalization of the chelate system was achieved by reacting the previously mono N-Boc-protected diamines or commercially available amino alkyl carboxylic acids with bis(imidazol-2-yl)nitromethane in the presence of aqueous NaOH (Scheme 2). Under these conditions, the nitro group is displaced by the nucleophilic amino group forming the corresponding NR-substituted 2,2′-bis(imidazolyl)methanes. Deprotection of the Boc group was accomplished in 3 M HCl at 50 °C (Scheme 3). The 1H/ 13C NMR and mass spectra and elemental analyses confirmed the composition and the symmetric structure of the ligands. In all compounds the four C-H protons of the imidazole rings are chemically equivalent, forming singlets around 7 ppm. Ligands 11 and 13 could be produced in two and three steps, respectively. The chelating moiety was built up by a double alkylation of the primary amino group with methyl bromoacetate in the presence of triethylamine in good yields. Subsequent saponification of the ester groups and/or deprotection of the N-Boc group afforded the bifunctional IDA derivatives (Scheme 3). The 1H/13C NMR

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Scheme 4

Figure 1. Labeling yield as a function of the ligand concentration in PBS buffer pH ) 6.5 (ligands 8/10) and pH ) 7.5 (ligands 11/13) 60 min, 60 °C.

and mass spectra and elemental analyses confirmed the composition and the structures of the ligands. Radiolabeling and in Vitro Stability. Radiolabeling of ligands 8/10 and 10/13 with precursor 1 was performed in PBS buffer at pH 6.5-7.5 (Scheme 4). Different labeling conditions have been tested. Short incubation at higher temperature (10 min, 100 °C) or longer incubation at lower temperature (37 °C, 3 h) revealed almost complete consumption of 1, but it was observed that under these conditions the reformation of perrhenate prohibits labeling yields better than 40% of the initial activity. The best conditions for radiolabeling were found to be 60 °C for 60 min in PBS. All tested ligand systems formed single species with the precursor 1. Plotting the labeling yields of the different chelate systems as a function of ligand concentration revealed steep sigmoid curves (Figure 1). Generally the 2,2′diimidazolylmethylamine chelates (8/10) yielded the corresponding Re-188 complexes at 1 order of magnitude lower concentration (5 × 10-5M) than the IDA-chelates (5 × 10-4M for 11/13). At these concentrations, labeling yields >95% in respect to [188Re(OH2)3(CO)3]+ and specific activities of up to 220 GBq/µmol ligand (based on initial activity of 14 GBq/mL 188ReO4-) could be achieved. The pendent additional functional groups (CO2H or NH2) did not seem to have an influence on the labeling yields. Stability of the Re-188 complexes was evaluated in human serum at 37 °C over a period of 48 h (Table 1). The 188Re(CO)3 complexes with ligands 8/10 showed generally a higher stability (80 ( 4% 24 h and 63 ( 3% 48 h postincubation in human serum) than those with ligands 11/13 (45 ( 10% 24 h and 34 ( 3% 48 h postincubation in human serum). The TLC and HPLC analyses of aliquots of the incubated plasma samples revealed beside the intact complexes decomposition predominantly to 188ReO4-. Only minor transchelation and aggregation to plasma proteins have been observed (>4% of total activity). Preparation and Analysis of Rhenium Complexes. To determine the coordinative features of the novel ligand systems, two representative rhenium model complexes with ligand 8b and 13a have been prepared and fully characterized including the X-ray structures. The complexes revealed almost identical retention times on the HPLC column ([Re(8b)(CO)3]+: 19.8 min; [Re(13a)(CO)3]: 20.4 min) as the corresponding radioactive complexes ([188Re(8b)(CO)3]+: 19.9 min; [188Re(13a)(CO)3]: 20.2 min) proving the identity of the complexes on the macroscopic and the nca level. Analysis of the 1H NMR

Table 1. Stability (%a) of 188Re(CO)3 Complexes with Ligands 8/10 and 11/13 in Human Serum Albumin at 37 °C time/ligand

1h

4h

24 h

48 h

8a 8c 8d 10a 10b 11a 11b 13a 13b

96 ( 1 95 ( 1 94 ( 6 98 ( 4 92 ( 5 90 ( 6 95 ( 6 92 ( 6 93 ( 8

95 ( 1 85 ( 6 92 ( 2 94 ( 3 81 ( 7 68 ( 4 62 ( 7 58 ( 6 68 ( 9

85 ( 5 80 ( 5 76 ( 5 83 ( 7 78 ( 4 50 ( 7 52 ( 2 48 ( 7 40 ( 14

63 ( 10 65 ( 3 61 ( 10 68 ( 6 60 ( 4 33 ( 5 38 ( 8 35 ( 12 30 ( 10

a

Values represent the means ( SD (n ) 3).

Figure 2. 2D proton NMR (COSY) of the aromatic region (imidazole protons) of complex [Re(8b)(CO)3]Br. For proton numbering, see Figure 3.

spectrum of complex [Re(8b)(CO)3]Br showed four doublets with an intensity of one proton each. Two-dimensional NMR experiments (1H/1H COSY) revealed coupling between two pairs of protons (Figure 2) indicating an unsymmetrical coordination or structure of the complex. A concluding explanation for this observation could be found after analysis of the X-ray structure. Crystals of X-ray quality could be grown by exchange of the bromide in complex [Re(8b)(CO)3]Br with the more bulky perchlorate anion. The ligand coordinates tridentately via the two imidazole rings and the secondary amino group

Radiolabeled Biomolecules for Therapeutic Application

Figure 3. ORTEP (18) of the complex cation [Re(8b)(CO)3]+. Re-C(13) 1.950(6) Å; Re-N(1) 2.194(6) Å; N(3)-C(8) 1.470(8) Å; N(3)-C(4) 1.522(7) Å; C(3)-C(4) 1.491(9) Å; C(4)-C(5) 1.518(8) Å; C(12)-Re-C(13) 89.8(3)°; C(12)-Re-N(1) 98.5(3)°; N(1)Re-N(4) 81.00(19)°; N(1)-Re-N(3) 74.93(19)°; N(4)-Re-N(3) 73.68(18)°; C(8)-N(3)-C(4) 115.1(4)°; C(3)-C(4)-C(5) 104.2(5)°.

as expected, forming two five-membered and one sixmembered ring with the metal center (Figure 3). The structure clearly reveals the asymmetry of the ligand/ complex after complexation. In respect to the N(3)-C(4) bond one of the imidazole rings is trans and one is cis oriented in respect to the acetyl methyl ester group (Figure 3). Clearly the orientation of the residue at the coordinated secondary amino group must be the reason for the chemical nonequivalence of the imidazole rings. The fact, that this NMR pattern was observed for all complexes with an NR-substituted 2,2′-diimidazolylmethane amine ligand but not with the unsubstituted is a further evidence for this assumption. Temperaturedependent NMR experiments (20-90 °C) did not show a significant change of these patterns, proving the rigid and inert coordination of the ligand to the metal center. The IR spectrum of compound [Re(8b)(CO)3]Br showed the typical fac-M(CO)3 pattern for the CO ligands at 2028 and 1922 cm-1/1904 cm-1. This is significantly blueshifted, compared to the educt (NEt4)2[ReBr3(CO)3] (2000 cm-1, 1868 cm-1). The neutral complex [Re(13aH+)(CO)3] crystallized directly as colorless needles from the reaction medium (H2O). Analysis of the 1H NMR spectrum of complex [Re(13aH+)(CO)3] revealed a typical pattern of an AB system for the four protons of the coordinated IDA moiety. The previously identical NCH2COO protons of the IDA moiety became nonequivalent upon rigid coordination to the metal center by virtue of their different chemical environment. The same spectroscopic features have been observed for all complexes with ligands 11 and 13 and were also reported for rhenium-tricarbonyl complexes of IDA-functionalized desoxyglucose and glucose derivatives (19). The IR spectrum of compound [Re(13aH+)(CO)3] showed M-CO stretch frequencies at 2018 and 1902 cm-1. The X-ray structure confirmed the tridentate coordination of the ligand via the two carboxylates and the tertiary amino group (Figure 4). CONCLUSION

The organometallic precursor [188Re(H2O)3(CO)3]+ can now be prepared in a simple one-step synthesis in good yields and high specific activities. These procedures will allow the easy performance of therapeutic studies using the organometallic labeling approach. In fact in vitro

Bioconjugate Chem., Vol. 13, No. 4, 2002 755

Figure 4. ORTEP (18) of the complex [Re(13aH+)(CO)3]. H-atoms are omitted for clarity. Re(1)-C(1) 1.919(4) Å; Re(1)O(4) 2.114(3) Å; Re(1)-O(6) 2.153(3) Å; Re(1)-N(1) 2.239(3) Å; C(2)-Re(1)-C(1) 87.74(17)°; C(3)-Re(1)-C(1) 88.40(17)°; C(2)Re(1)-O(4) 173.96(13)°; O(4)-Re(1)-O(6) 78.19(11)°; O(4)-Re(1)-N(1) 78.69(11)°; O(6)-Re(1)-N(1) 76.78(11)°.

experiments with Re-188 tricarbonyl-labeled antibodies against bladder cancer have shown the superior characteristic of the carbonyl technology compared to “classical” Re(V) methods (20). The novel, tridentate, bifunctional chelating systems presented in this work are versatile and offer the possibility to insert presumably any type (e.g. alkyl, poly(ethylene glycol), benzene) and length of spacer adaptable to different biomolecules. Particularly with ligands containing a bis(imidazole-2-yl)methylamine moiety high labeling yields should be possible at low ligand concentration. ACKNOWLEDGMENT

We thank Judith Stahel, for her assistance in preparing the Re-188 complexes and Prof. Horst Bruchertseifer and Dr. Bernd Ja¨ckel for generously providing Na[188ReO4]. This work was supported by Mallinckrodt-Tyco Inc. Supporting Information Available: Tables of crystal data, structure solution and refinement, atomic coordinates, bond lengths and angles and anisotropic thermal parameters for [Re(8b)(CO)3]ClO4 and [Re(13aH+)(CO)3] in CIF-format. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Volkert, W. A., and Hoffman, T. J. (1999) Therapeutic Radiopharmaceuticals. Chem. Rev. 99, 2269-2292. (2) Liu, S., and Edwards, E. S. (2001) Bifunctional Chelators for Therapeutic Lanthanide Radiopharmaceuticals. Bioconjugate Chem. 12, 7-34. (3) Coursey, B. M., J. M., C., Cessa, J., Hoppes, D. D., Schima, F. J., Unterweger, M. P., Golas, D. B., Callahan, A. P., Mirzadeh, S., and Knapp, F. F. (1990) Assay of the Eluent from the Alumina-Based Tungsten-188-Rhenium-188 Generator. Radioact. Radiochem. 38-49. (4) Ferro-Flores, G., Pimentel Gonzalez, G., Gonzales-Zavala, M. A., Artega de Murphy, C., Melendez Alafort, L., Tendilla, J. I., and Croft, B. Y. (1999) Preparation, Biodistribution, and Dosimetry of 188Re Labeled MoAb or cea1 and Its F(ab′)2 Fragments by Avidin-Biotin Strategy. Nucl. Med. Biol. 26, 57-62. (5) Karacay, H., McBride, W. J., Griffiths, G. L., Sharkley, R. M., Barbet, J., Hansen, H. J., and Goldenberg, D. M. (2000) Experimental Pretargeting Studies of Cancer with Humanized anti-CEAXMurine anti-[In-DTPA] Bispecific Antibody Constructs and a 99mTc-/188Re-Labeled Peptide. Bioconjugate Chem. 11, 842-854.

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