Mixed-Ligand Rhenium-188 Complexes with Tetradentate

Bioconjugate Chem. 2005, 16, 634−643

634

Mixed-Ligand Rhenium-188 Complexes with Tetradentate/ Monodentate NS3/P (‘4 + 1’) Coordination: Relation of Structure with Antioxidation Stability Eik Schiller,† Sepp Seifert,† Francesco Tisato,‡ Fiorenzo Refosco,‡ Werner Kraus,§ Hartmut Spies,† and Hans-Juergen Pietzsch*,† Forschungszentrum Rossendorf, Institut fu¨r Bioanorganische und Radiopharmazeutische Chemie, 510119, D-01314 Dresden, Germany, Consiglio Nazionale delle Ricerche, Istituto di Chimica Inorganica e delle Superfici, Corso Stati Uniti 4, I-35127 Padova, Italy, and Bundesanstalt fu¨r Materialforschung und -pruefung, Richard-Willstaetter-Strasse 11, D-12489 Berlin, Germany. Received October 26, 2004; Revised Manuscript Received February 10, 2005

Development of new radiopharmaceuticals based on rhenium-188 depends on finding appropriate ligands able to give complexes with high in vivo stability. Rhenium(III) mixed-ligand complexes with tetradentate/monodentate (‘4 + 1’) coordination of the general formula [Re(NS3)(PRR′R′′)] (NS3 ) tris(2-mercaptoethyl)amine and derivatives thereof, PRR′R′′ ) phosphorus(III) ligands) appear to be among the promising tools to achieve this goal. According to this approach, we synthesized and characterized a series of rhenium model complexes. In vitro stabilities of the corresponding rhenium-188 complexes were determined by incubating 2-3 MBq or alternatively 37 MBq of the complexes in phosphate buffer, human plasma, and rat plasma, respectively, at 22 °C or 37 °C, followed by checking the amount of 188ReO4- formed after 1 h, 24, and 48 h by thin-layer chromatography. The rate of perrhenate formation varied over a wide range, depending primarily on the nature of the phosphorus(III) ligand. Physicochemical parameters of the corresponding nonradioactive rhenium complexes were analyzed in detail to find out the factors influencing their different stability and furthermore to design new substitution-inert ‘4 + 1’ complexes. Tolman’s cone angle of phosphorus(III) ligands and the lipophilic character of the inner coordination sphere were found to be crucial factors to build up stable rhenium ‘4 + 1’ complexes. Additional information useful to describe electronic and steric properties of these compounds were selected from electronic spectra (wavelength of the RefS charge-transfer band), cyclovoltammetric measurements (E° of the ReIII/ReIV couple), and NMR investigations (31P chemical shift of coordinated P(III) ligands).

INTRODUCTION

Rhenium-188 is an attractive candidate for therapeutic applications in oncology due to its nuclide properties (Eβmax ) 2.1 MeV, t1/2 ) 16.9 h) and its daily availability from a 188W/188Re generator (1-3). To exploit the potencies of this nuclide, target-specific radiotherapeuticals must be developed to accumulate the radioactivity in the tumor and to spare healthy tissue from radiation. This can be achieved by linking a radionuclide-bearing chelate to a target-seeking biomolecule. Being capable to control the biodistribution requires kinetically inert and metabolically stable complexes. Despite some rhenium-188 tricarbonyl complexes with tridentate ligands (4) and in some cases 188Re(O)MAG3 that show fairly good stability both in vitro and in vivo (5-7), antioxidation stability at high level of radioactivity remains one of the most important questions in rhenium radiopharmacology. A promising approach for attaching rhenium-188 to biomolecules is the so-called ‘4 + 1’ mixed ligand system (8). Here a rhenium(III) center is coordinated by the tetradentate tris(2-mercaptoethyl)amine chelator (NS3) * To whom correspondence should be addressed. Fax: +49 351 260 3232. E-mail: [email protected]. † Institut fu ¨ r Bioanorganische und Radiopharmazeutische Chemie. ‡ Istituto di Chimica Inorganica e delle Superfici. § Bundesanstalt fu ¨ r Materialforschung und -pruefung.

Figure 1. The ‘4 + 1’ mixed ligand approach.

or a derivative thereof and a phosphorus(III) compound or an isocyanide as monodentate ligand. The latter also serves as a linker for coupling target-specific biomolecules such as peptides, antibodies, and oligonucleotides (Figure 1). Until now, ‘4 + 1’ complexes of technetium and rhenium with the NS3 chelator tris(2-mercaptoethyl)amine and several isocyano or phosphine ligands were predominantly of lipophilic character (9, 10). Development of more hydrophilic rhenium complexes may help to minimize exposure of radiation-sensitive organs by fast excretion of radioactive metabolites and furthermore enables radiolabeling of water-soluble biomolecules such as antibodies and peptides in aqueous solution. A first attempt to produce more hydrophilic ‘4 + 1’ complexes was published recently by our group, involving the preparation of a tetradentate NS3 ligand bearing a carboxyl group (11). In the present work we report on the extension of the ‘4 + 1’ system using different monodentate phosphorus-

10.1021/bc049745a CCC: $30.25 © 2005 American Chemical Society Published on Web 04/14/2005

Mixed-Ligand Rhenium-188 Complexes

Bioconjugate Chem., Vol. 16, No. 3, 2005 635

Scheme 1. Reaction Routes to Re(III) ‘4 + 1’ Complexes

(III) ligands, including both lipophilic and hydrophilic tertiary phosphines and phosphites (see Scheme 1). Moreover, the use of the unsubstituted NS3 chelator compared with substituted NS3 ligands bearing a carboxyl group (NS3COOH) and an isopropyl amide (NS3CONHiPr), respectively, offers the possibility of finetuning the system. We evaluated the potentials of the resulting complexes as tools for therapeutic purposes. Therefore, in vitro stabilities, which we defined as the amount of perrhenate formed after incubation of the rhenium-188 complexes at low (2-3 MBq) or medium (37 MBq) activity level in phosphate buffer, human plasma, and rat plasma, were determined. Physicochemical parameters of the corresponding nonradioactive rhenium complexes were analyzed in detail to find out trends useful to explain the different in vitro stabilities of various rhenium-188 complexes. Results of this analysis were used to design complexes of superior in vitro stability. EXPERIMENTAL PROCEDURES

General. All solvents and commercially available substances were of reagent grade and used without further purification. Commercially available phosphines and phosphites are dimethylphenylphosphine 1 and 4-(diphenylphosphino)benzoic acid 5 (Aldrich), tris(2cyanoethyl)phosphine 3 and tris(o-tolyl)phosphite 6 (Alfa Aesar). Diphenylphosphino acetic acid 4 was synthesized according to the method described by Jarolim et al. (12). Syntheses of the tetradentate ligand tris(2-mercaptoethyl)amine (NS3) and rhenium complex Re1 were reported elsewhere (8). Tris(2-mercaptoethyl)amine was isolated and applied as the oxalate. 2-[Bis(2-mercaptoethyl)amino]-3-mercaptopropionic acid (NS3COOH) was prepared as hydrochloride salt by double N-alkylation of S-benzylcysteine (11). 188ReO4- was eluted from a 188W/ 188Re generator (Oak Ridge National Laboratories, Oak Ridge, TN). Instrumentation. Nuclear magnetic resonance spectra were recorded on a 400 MHz Varian Inova 400 spectrometer. The 1H and 13C chemical shifts are reported relative to residual solvent signals or TMS as reference. 31 P chemical shifts are reported relative to H3PO4 as external reference. Mass spectrometric measurements

were performed with a Micromass Tandem Quadropole Mass Spectrometer (Quadro LC) operating in the MS mode. Mass spectral data were recorded in the positive ESI mode. About 10-4 mol of the sample dissolved in 1.0 mL of methanol or acetonitrile were injected at a flow rate of 5 µL/min. Elemental analyses were performed on a LECO Elemental Analyzer CHNS-932. Cyclic voltammetry was carried out in dichloromethane solutions (1 × 10-3 mol dm-3) with [n-Bu4N][ClO4] (0.1 mol dm-3) as supporting electrolyte, at a stationary platinum-disk electrode (area ca. 1.28 mm2), which was cleaned after each run, with scan rate 0.2 V s-1 at 293 K. Potentials were measured relative to an Ag-wire pseudoelectrode using the Fc/Fc+ couple as internal reference. Controlled potential coloumetries of dichloromethane solutions were performed using an Amel model 721 integrator, in a H-shaped cell containing, in arm 1, a platinum-gauzed working electrode, and an Ag/Ag+ reference isolated inside a salt bridge by a medium glass frit and, in arm 2, an auxiliary platinum-foil electrode. UV/vis spectra were recorded on a SPECORD S10 spectrometer (Carl Zeiss Jena). HPLC analysis and purification of rhenium-188 complexes were performed using a Perkin-Elmer instrument consisting of a Turbo LC System with a quaternary pump (Series 200 LC Pump) and a programmable absorbance detector model 785A. Nonradioactive rhenium complexes were monitored by their UV absorption at 254 nm, and rhenium-188 complexes by γ-ray detection (Bohrloch, NaI(Tl) crystal). Synthesis of {[2-(Diethylamino)ethyl]phosphinediyl]}dimethanol 2. To a solution of 0.001 mol of diethyl [2-(diethylamino)ethyl]phosphonate, synthesized according to the method of Kosolapoff et al. (13), in 5 mL of dry diethyl ether, at 0 °C, was added 1.5 mL of a 1 mol/L LiAlH4 solution (diethyl ether) dropwise via syringe. After the mixture was stirred at 0 °C under an argon atmosphere for 0.5 h about 3 mL of H2O was added. The organic layer was separated, and the aqueous layer was extracted two times each with 3 mL of diethyl ether. The solvent of the combined organic solutions was removed in an argon stream. To the remaining oil were added 3 mL of degassed EtOH, 250 µL (0.003 mol) of a formaldehyde solution (37% in H2O/MeOH), and 0.5 mL of 3 N

636 Bioconjugate Chem., Vol. 16, No. 3, 2005

aq HCl. After being stirring at room temperature overnight, the solvent was stripped in vacuo, yielding 88% of a colorless viscous oil. Phosphine 2 was liberated in situ during the complex synthesis step. General Synthesis Procedure for the Preparation of Rhenium Complexes Re3, Re4, Re6. To a solution of 0.1 mmol of tris(2-mercaptoethyl)ammonium oxalate (NS3) and 0.3 mmol of the phosphine/phosphite in 5 mL of methanol (for Re3) or acetonitrile (for Re4 and Re6) was added 0.1 mmol (26.8 mg) of NH4ReO4 (dissolved in ca. 200 µL of concentrated HCl for Re3 and Re4). The reaction mixture was refluxed under an argon atmosphere for 2 h. The solvent was removed in vacuo and the residue purified by column chromatography (silica). Eluents were chloroform/ethyl acetate (90:10) for Re3, chloroform/ethyl acetate/TFA (90:10:0.1) for Re4, and n-hexane/ethyl acetate (50:50) for Re6. Crystals suitable for X-ray structure analysis were obtained by slow solvent evaporation of chloroform solutions of Re3, Re4, or Re6. Re3. Yield: 64% dark-green crystals. Mp: >210 °C (decomp). Anal. (C15H24N4S3PRe) Calcd: C 31.40%, H 4.22%, N 9.77%, S 16.76%. Found: C 31.01%, H 4.36%, N 9.72%, S 16.77%. UV/vis (CH2Cl2): λmax(log ) ) 344 (3.0), 450 (3.1), 577 (2.4). 1H NMR (DMSO-d6): 2.29 (m, 6 H, PCH2), 2.61 (m, 6 H, CH2CN), 2.90 (bs, 12 H). 13C NMR (DMSO-d6) 13.50 (d, J ) 6.10 Hz, 3 C, CH2CN), 31.60 (d, J ) 26.70 Hz, 3 C, PCH2), 46.60 (d, J ) 5.34 Hz, 3 C, SCH2), 59.36 (s, 3 C, NCH2), 121.55 (s, 3 C, CN). 31P NMR (DMSO-d ) 18.42 (s). ESI-MS (m/z): 574 [M]+. 6 Re4. Yield: 34% dark-green crystals. Mp: >215 °C (decomp). Anal. (C20H25NO2S3PRe) Calcd: C 38.45%, H 4.03%, N 2.24%, S 15.39%. Found: C 37.90%, H 4.13%, N 1.92%, S 13.84%. UV/vis (CH2Cl2): λmax(log ) ) 455 (2.9), 590 (2.2). 1H NMR (CDCl3) 2.80 (bs, 12 H), 3.64 (d, J ) 7.87 Hz, 2 H, CH2), 7.17 (m, 2 Harom.), 7.24 (m, 4 Harom.), 7.62 (m, 4 Harom.). 13C NMR (CDCl3) 46.38 (d, J ) 4.58 Hz, 3 C, SCH2), 48.86 (d, J ) 16.79 Hz, 1 C, PCH2), 60.00 (s, 3 C, NCH2), 127.51 (d, J ) 9.92 Hz, 4 Carom.), 128.99 (s, 2 Carom.), 133.06 (d, J ) 11.44 Hz, 4 Carom.), 144.03 (d, J ) 45.78 Hz, 2 Carom.), 172.47 (s, 1 C, C(O)). 31 P NMR (CDCl3) 18.20 (s). Re6. Yield: 24% brownish-red crystals. Mp: 225-233 °C. Anal. (C27H33NO3S3PRe) Calcd: C 44.25%, H 4.54%, N 1.91%, S 13.12%. Found: C 44.16%, H 4.53%, N 1.82%, S 13.24%. UV/vis (CH2Cl2): λmax(log ) ) 356 (3.0), 418 (3.2), 524 (2.4). 1H NMR (CDCl3) 2.17 (s, 9 H, CH3), 2.91 (bs, 12 H), 6.95 (m, 3 Harom.), 7.05 (m, 3 Harom.), 7.11 (d, J ) 7.32 Hz, 3 Harom.), 7.55 (d, J ) 8.06 Hz, 3 Harom.). 13C NMR (CDCl3) 17.22 (s, 9 C, CH3), 44.08 (d, J ) 6.10 Hz, 3 C, SCH2), 59.06 (s, 3 C, NCH2), 121.40 (d, J ) 3.82 Hz, 3 Carom.), 123.33 (s, 3 Carom.), 126.42 (s, 3 Carom.), 130.65 (d, J ) 4.58 Hz, 3 Carom.), 130.80 (s, 3 Carom.), 151.99 (d, J ) 8.39 Hz, 3 Carom.). 31P NMR (CDCl3) 115.54 (s). ESIMS (m/z): 733 [M]+. General Synthesis Procedure for the Preparation of Re2, Re5, Re7, Re8. The rhenium precursor [Re(tu)6]Cl3 × H2O was synthesized by the method described by Gambino et al. (14). The monodentate ligand, tris(2mercaptoethyl)ammonium oxalate (NS3) (0.1 mmol, 28.6 mg), and [Re(tu)6]Cl3 × H2O (0.1 mmol, 76.7 mg) were dissolved in 10 mL of methanol. For the synthesis of Re2, 100 µL triethylamine was added to the mixture to convert the phosphonium salt into the corresponding tertiary phosphine 2. The solution was refluxed under an argon atmosphere for 3 h. After being cooling to room temperature, the reaction mixture was filtered and the solvent stripped in vacuo. Re2 was purified by column chromatography (silica, acetone/chloroform/triethylamine (66:33:

Schiller et al.

0.1)). In the case of Re5, Re7, and Re8, the crude product, which was impured by its methyl ester derivatives, was dissolved in a mixture of 3 mL of dioxane and 1 mL of 1 N NaOH and refluxed for 3 h. The reaction mixture was then acidified with 0.1 N HCl and the product extracted with chloroform. After evaporation of the solvent, the complex was further purified by silica column chromatography (n-hexane/ethyl acetate/TFA (50: 50:0.1). Crystals of Re2 suitable for X-ray structure analysis were obtained by slow solvent evaporation of a solution of Re2 in methylenchloride/n-hexane. Re2. Yield: 31% dark-green crystals. Mp: 159-162 °C. UV/vis (CH2Cl2): λmax(log ) ) 340 (2.9), 461 (3.0), 592 (2.2). 1H NMR (CDCl3) 1.19 (t, J ) 7.33 Hz, 6 H, CH3), 2.59 (m, 2 H, PCH2CH2), 2.87 (q, J ) 7.33 Hz, 4 H, NCH2CH3), 2.90 (bs, 12 H), 3.06 (m, 2 H, NCH2CH2), 4.22 (m, 4 H, CH2OH). 13C NMR (CDCl3) 8.90 (s, 2 C, CH3), 21.64 (d, J ) 26.70 Hz, 1 C, PCH2, 46.18 (s, 2 C, NCH2), 47.32 (d, J ) 4.58 Hz, 3 C, SCH2), 50.21 (d, J ) 12.17 Hz, 1 C, NCH2), 59.74 (s, 3 C, NCH2), 73.17 (d, J ) 28.23 Hz, 2 C, CH2OH). 31P NMR (CDCl3) 16.50 (s). ESI-MS (m/z): 575 [M + H]+. Re5. Yield: 25% dark-green crystals. Mp: >120 °C (decomp). UV/vis (CH2Cl2): λmax(log ) ) 457 (3.3), 591 (2.6). 1H NMR (CDCl3) 2.87 (bs, 12 H), 7.28 (m, 6 Harom.), 7.45 (m, 4 Harom.), 7.53 (d, J ) 7.51 Hz, 2 Harom.), 7.98 (d, J ) 8.24 Hz, 2 Harom.). 13C NMR (CDCl3) 47.12 (s, 3 C, SCH2), 60.54 (s, 3 C, NCH2), 128.04 (d, J ) 6.87 Hz, 4 Carom.),129.15 (s, 1 Carom.),129.42 (m, 4 Carom.), 134.02 (d, J ) 9.16 Hz, 2 Carom.), 134.46 (d, J ) 9.15 Hz, 4 Carom.), 143.31 (d, J ) 47.30 Hz, 2 Carom.), 151.56 (d, J ) 54.93 Hz, 1 Carom.), 171.00 (s, 1 C, C(O)). 31P NMR (CDCl3) 30.68 (s). Re7. Yield: 36% dark-green crystals. Mp: >145 °C (decomp). UV/vis (CH2Cl2): λmax(log ) ) 455 (3.0), 586 (2.2). 1H NMR (CDCl3) 2.47 (m, 1 H), 2.76 (m, 3 H), 3.09 (m, 2 H), 3.22 (m, 1 H), 3.40 (dd, J ) 13.64, 4.85 Hz, 1 H), 3.51 (m, 1 H), 3.63 (m, 2 H), 7.30 (m, 6 Harom.), 7.44 (m, 6 Harom.), 7.93 (dd, J ) 8.24, 1.28 Hz, 2 Harom.). 13C NMR (CDCl3) 46.46 (d, J ) 3.05 Hz, 1 C, SCH2), 48.96 (d, J ) 5.34 Hz, 1 C, SCH2), 50.53 (d, J ) 4.58 Hz, 1 C, SCH2), 55.85 (s, 1 C, NCH2), 57.25 (s, 1 C, NCH2), 70.44 (s, 1 C, NCH), 128.13 (d, J ) 9.92 Hz, 4 Carom.), 129.05 (d, J ) 1.53 Hz, 1 Carom.), 129.55 (m, 4 Carom.), 134.10 (d, J ) 11.44 Hz, 2 Carom.), 134.47 (d, J ) 11.44 Hz, 4 Carom.), 142.69 (d, J ) 47.30 Hz, 2 Carom.), 151.83 (d, J ) 41.96 Hz, 1 Carom.), 171.82 (s, 1 C, C(O)), 175.23 (s, 1 C, C(O)). 31P NMR (CDCl ) 29.94 (s). 3 Re8. Yield: 29% dark-green crystals. Mp: >155 °C (decomp). UV/vis (CH2Cl2): λmax(log ) ) 457 (3.2), 590 (2.4). 1H NMR (CDCl3) 1.16 (d, J ) 6.59 Hz, 3 H, CH3), 1.22 (d, J ) 6.59 Hz, 3 H, CH3), 2.51 (m, 1 H), 2.81 (m, 3 H), 3.22 (m, 5 H), 3.44 (m, 1 H), 3.78 (m, 1 H), 4.15 (m, 1 H), 5.52 (m, 1 H, NH), 7.35 (m, 6 Harom.), 7.47 (m, 4 Harom.), 7.55 (dd, J ) 9.98, 8.52 Hz, 2 Harom.), 8.02 (dd, J ) 8.15, 1.37 Hz, 2 Harom.). 13C NMR (CDCl3) 22.66 (s, 1C, CH3), 23.23 (s, 1C, CH3), 42.53 (s, 1 C, CH(CH3)2), 57.00 (m), 73.50 (m), 128.15 (s), 129.20 (s), 129.56 (s), 134.17 (s), 134.57 (s), 168.35 (s, 1 C, C(O)), 171.35 (s, 1 C, C(O)). 31 P NMR (CDCl3) 29.85 (s). ESI-MS (m/z): 772 [M]+. X-ray Data Collection and Processing. The X-ray data were collected at room temperature (293 K) on a SMART-CCD diffractometer (SIEMENS), using graphite monochromatized Mo KR radiation (λ ) 0.71073 Å). A summary of the crystallographic data of Re2, Re3, Re4, and Re6 is given in Table 1. The positions of the nonhydrogen atoms were determined by the heavy atom technique. After anisotropic refinement of these positions,



Table 1. Crystallographic Data of X-ray Diffraction Studies of Rhenium Complexes Re2, Re3, Re4, and Re6 formula formula wt crystal system space group a (Å) b (Å) c (Å) R (deg) β (deg) χ (deg) volume (Å3) Z T (K) F (g/cm3) absorption coeff (mm-1) F(000) λ (Mo KR) (Å) crystal size (mm3) θ-range from data collection index ranges reflections collected independent reflections goodness-of fit of F2 R [I > 2σ (I)] R (all data)

Re2

Re3

Re4

Re6

C14H32N2O2PReS3 573.77 monoclinic P2(1)/c 10.646(6) 12.198(7) 16.203(10) 90.00 91.699(12) 90.00 2103(2) 4 273(2) 1.812 6.160 1136 0.71073 0.8 × 0.024 × 0.016 1.91-25.00 -12 e h e 12 -14 e k e 14 -12 e l e 19 10212 3703 0.634 R1 ) 0.0398 wR2 ) 0.0950 R1 ) 0.0778 wR2 ) 0.1098

C15H24N4PReS3 573.73 monoclinic P2(1)/c 22.313(8) 12.791(4) 14.924(5) 90.00 97.063(7) 90.00 4227(2) 8 273(2) 1.803 6.126 2240 0.71073 0.25 × 0.07 × 0.015 0.92-25.00 -26 e h e 21 -14 e k e 15 -17 e l e 17 20443 7421 0.977 R1 ) 0.0587 wR2 ) 0.1205 R1 ) 0.1503 wR2 ) 0.1393

C20H25NO2PReS3 624.78 orthorhombic Pbca 14.436(18) 12.568(16) 29.67(3) 90.00 90.00 90.00 5383(12) 8 273(2) 1.836 5.124 2912 0.71073 0.85 × 0.5 × 0.15 1.97-25.00 -17 e h e 9 -14 e k e 14 -37 e l e 27 24134 4740 0.877 R1 ) 0.0555 wR2 ) 0.1202 R1 ) 0.1094 wR2 ) 0.1410

C27H33NO3PReS3 732.89 monoclinic P2(1)/n 9.716(4) 12.182(5) 24.439(10) 90.00 100.508(8) 90.00 2844(2) 4 273(2) 1.712 4.578 1456 0.71073 0.5 × 0.25 × 0.2 2.38-29.00 -12 e h e 12 -16 e k e 9 -31 e l e 32 17707 7195 0.974 R1 ) 0.0388 wR2 ) 0.0958 R1 ) 0.0501 wR2 ) 0.0983

Table 2. Selected Bond Lengths (Å) and Angles (deg) of Re2, Re3, Re4, and Re6 Re-N(1) Re-S(1) Re-S(2) Re-S(3) Re-P N(1)-Re-S(1) N(1)-Re-S(2) N(1)-Re-S(3) S(1)-Re-S(2) S(1)-Re-S(3) S(2)-Re-S(3) N(1)-Re-P S(1)-Re-P S(2)-Re-P S(3)-Re-P

Re2

Re3

Re4

Re6

2.207(7) 2.226(3) 2.230(3) 2.234(3) 2.297(2) 85.5(2) 84.5(2) 85.6(2) 119.00(11) 118.95(11) 119.98(12) 177.9(2) 93.24(9) 97.59(8) 93.53(9)

2.202(11) 2.235(4) 2.212(4) 2.227(4) 2.291(4) 85.6(3) 84.9(4) 85.8(3) 118.71(19) 118.50(19) 120.92(19) 178.7(3) 93.21(15) 95.18(15) 95.23(14)

2.179(10) 2.257(4) 2.252(4) 2.260(4) 2.301(4) 86.4(2) 85.1(2) 84.2(2) 118.60(12) 119.81(14) 119.56(12) 177.4(2) 94.63(11) 96.42(11) 93.23(12)

2.207(4) 2.2292(14) 2.2321(14) 2.2317(13) 2.2228(13) 85.17(9) 85.03(11) 85.98(10) 119.12(5) 119.09(6) 119.88(5) 177.84(9) 92.75(4) 95.51(6) 95.54(4)

the hydrogen positions were calculated according to ideal geometries. Empirical absorption corrections were made using psi scans. Most of the calculations were carried out in the SHELXTL system with some local modifications. Selected bond lengths and angles are reported in Table 2. CCDC 253461 (Re2), CCDC 253462 (Re3), CCDC 253463 (Re4), and CCDC 253464 (Re6) 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 emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. Synthesis Procedure for 188Re Complexes. Synthesis was carried out via the EDTA intermediate as previously described by Seifert and co-workers (11). 188Re-EDTA. 3-5 mL of perrhenate eluate from a 188W/ 188 Re generator were added together with 0.5 mL of 0.1 N HCl to a kit vial containing 5 mg of EDTA, 5 mg of mannitol, and 1.0 mg of SnCl2 in freeze-dried form under nitrogen. After 20-30 min at room temperature the complex formation is finished. Yield: >95% (TLC).

‘4 + 1’ 188Re Complexes. A 0.3 mg amount of the appropriate NS3 derivative and 0.05-0.20 mg of phosphine, dissolved in a mixture of 0.5 mL of water and 0.5 mL of methanol, were added with a syringe to the 188ReEDTA preparation, and the solution was heated at 50 °C (Re4, Re5, Re6) or 75 °C (Re1, Re2, Re3, Re7, Re8) for 30 min. Radiochemical yield: 50-90% (TLC), except Re6: 20-30%. The preparations were purified for stability studies by HPLC using the following methods. Method 1 (Re1, Re2, Re3, Re5): Hypersil ODS (10 µm, 250 × 8 mm, Knauer), 3 mL/min; solvent A: methanol; solvent B: 0.01 M phosphate buffer pH 7.4 (PBS); gradient elution: 0-10 min 0 to 100% A (Re1, Re3) or 0-10 min 30 to 100% A (Re2, Re5). Method 2 (Re4, Re7, Re8): PRP-1 (10 µm, 250 × 10 mm, Hamilton); 3 mL/min; solvent A: acetonitrile + 0.1% trifluoro acetic acid (TFA); solvent B: water + 0.1% TFA; gradient elution: 0-10 min 30 to 100% A. Method 3 (Re6): Zorbax 300 SB-C18 (5 µm, 250 × 9.4 mm, Agilent); 3 mL/min; solvent A: acetonitrile + 0.1% TFA; solvent B: water + 0.1% TFA; gradient elution: 0-10 min 30 to 100% A. Thin-layer chromatography (TLC) was performed using RP-18 silica gel strips (Merck) and methanol/0.01 M phosphate buffer pH 7.4 (90:10) for Re1-Re3 or acetonitrile/water/TFA (80:20:0.1) for Re4-Re8 as mobile phases. All compounds show Rf values between 0.4 and 0.6. The strips were scanned with a raytest Rita radioanalyzer. Stability Studies. The in vitro stability was determined by adding 2-3 MBq or 37 MBq of the relevant pure 188Re complex, dissolved in 100 µL of propylene glycol, to (i) 400 µL of phosphate buffer (PBS) pH ) 7.4; (ii) 200 µL of human plasma + 200 µL of phosphate buffer (PBS) pH ) 7.4; (iii) 200 µL of rat plasma + 200 µL of phosphate buffer (PBS) pH ) 7.4. The samples containing 2-3 MBq were incubated at room temperature (22 °C), the 37 MBq samples at 37 °C. All compounds were investigated at low activity level due to the low radiation exposure and easy performance

638 Bioconjugate Chem., Vol. 16, No. 3, 2005 Table 3. In Vitro Stabilities of

188Re

Schiller et al.

Complexes Re1-Re8 (%

phosphate buffer Re1 Re2 Re3 Re4 Re5 Re6 Re7 Re8

188ReO -, 4

values are presented as mean ( SD, n ) 3)

human plasma

rat plasma

1h

24 h

48 h

1h

24 h

48 h

1h

24 h

48 h

100 ( 0 65 ( 7 11 ( 4 1(1 3(0 1(1 7(1 4(2

100 ( 0 100 ( 0 49 ( 1 15 ( 1 3(1 10 ( 3 16 ( 2 8(2

100 ( 0 100 ( 0 67 ( 2 32 ( 6 4(1 17 ( 8 16 ( 1 10 ( 1

100 ( 0 84 ( 2 5(1 1(1 3(1 0(0 8(1 1(1

100 ( 0 100 ( 0 15 ( 1 27 ( 3 6(1 0(1 13 ( 2 2(1

100 ( 0 100 ( 0 22 ( 3 33 ( 8 6(0 0(0 13 ( 3 3(1

100 ( 0 100 ( 0 16 ( 2 1(1 3(1 2(1 6(2 2(1

100 ( 0 100 ( 0 33 ( 3 28 ( 4 7(2 9(2 9(2 3(0

100 ( 0 100 ( 0 42 ( 2 30 ( 5 9(4 26 ( 3 9(3 3(0

of the experiments. Only stable complexes have been investigated under harsher conditions. We chose the same conditions as those published by Schibli et al. (4), enabling the comparison of the results.

Table 4. Physicochemical Parameters vs in Vitro Stability of Complexes Re1-Re8

RESULTS

Re1 Re2 Re3 Re4 Re5 Re6 Re7 Re8

Rhenium Chemistry. Synthesis. As outlined in Scheme 1, the nonradioactive Re(III) ‘4 + 1’ model complexes can be prepared using two different methods: (A) reduction-substitution reactions starting from ammonium perrhenate (8) or (B) ligand-exchange reactions starting from the hexakisthiourea Re(III) precursor. In the first case, an equimolar amount of NS3 and an excess of phosphorus(III) ligand, dissolved in methanol (or acetonitrile for carboxyl group-bearing ligands and phosphites), react under reflux (2 h) with an acidic solution of perrhenate (without addition of HCl for phosphite ligand 6) under an argon atmosphere to afford stable Re(III) ‘4 + 1’ complexes. Alternatively, equimolar amounts of [Re(tu)6]Cl3, NS3 and phosphine react in refluxing methanol to give analogous Re(III) ‘4 + 1’ complexes in moderate yields. Both procedures are characterized by a typical change in color from colorless perrhenate to green or reddish, indicating the reduction from Re(VII) to Re(III) (method (A)), or from brownish to yellow-green in the case of ligand-exchange reactions (method (B)). Characterization. The Re(III) complexes have been characterized by means of (i) elemental analyses, which are in good agreement with the proposed formulation, (ii) cyclic voltammetry, (iii) spectroscopic measurements, comprising UV/vis and multinuclear NMR, (iv) mass spectrometry, and (v) X-ray structure analyses of four representative complexes including unsubstituted NS3 and phosphines 2, 3, or 4 and phosphite 6 (complexes Re2, Re3, Re4, and Re6). The diamagnetism shown by this class of Re(III) complexes is in agreement with low-spin d4 trigonal bipyramidal configuration (vide infra, X-ray description). Thus, NMR spectra of Re1-8 exhibit a sharp 31P singlet, which moves significantly downfield, by 30-40 ppm, upon coordination in phosphine containing complexes Re1-5 and Re7-8 compared to the values shown by uncoordinated ligands (see Table 4). On the contrary, such a singlet moves upfield by 16 ppm in the unique case of the coordinated phosphite in Re6. 1H and 13C spectra of Re(III) ‘4 + 1’ complexes show signals both in the aromatic and in the aliphatic regions, consistent with the formulation. In particular, proton spectra of complexes comprising the unsubstituted NS3 ligand (Re16) are dominated by a broad signal at δ 2.9 ppm, characteristic for the NS3 ethylene arms. Incorporation of a carboxyl group or a substituted carboxyl function in the NS3 framework causes such a signal to become a series of multiplets falling in the 2.4-3.7 ppm region (complexes Re7 and Re8; see Figure 2).

cone angle θ of the free P(III) ligand,a deg

E° [V]

122 ∼115 ∼140c ∼140 ∼145 141 ∼145 ∼145

-0.05 +0.04 +0.19 -0.02 +0.05 +0.37 +0.13 +0.04

% 188ReO4- in δ 31Pcomplex human plasma λ after 24 h [nm]b [ppm] 464 461 450 455 457 418 455 457

-13 +16 +20 +18 +31 +116 +30 +30

100 ( 0 100 ( 0 15 ( 1 27 ( 3 6(1 0(0 13 ( 2 2(1

a Taken or estimated from ref 18. b Most intensive band in the vis region. c Data estimated from Rahman (19) who corrected the 132° value reported by Tolman (18).

Figure 2. 1H NMR spectra of Re5 (above) and Re7 (below) in the region 2.2-3.7 ppm.

The UV/vis spectra of Re1-8 are characterized by intense absorption bands at approximately 420-460 nm ( approximately 1000), and less intense absorptions ( approximately 250) in the 520-590 nm region. The energy associated with these absorptions indicates that the former bands correspond to metal to ligand (RefS) charge-transfer transitions, whereas less intense bands correspond to d-d transitions. Both absorption bands are responsible for the green or reddish color of these complexes and move to shorter wavelengths in the phosphite-containing complex Re6 (see Table 4), indicating that the energy involved in the transfer for Re6 is higher compared to the energy implicated in phosphine complexes. The molecular structure of four representative examples of five-coordinate Re(III) ‘4 + 1’ complexes, Re2, Re3, Re4, and Re6, were investigated in the solid state by X-ray diffraction methods. A summary of the crystallographic data is given in Table 1, and selected bond lengths and angles are reported in Table 2. As outlined in Figures 3-6, the four complexes adopt a trigonalbipyramidal geometry. The three NS3 thiolate sulfurs form the trigonal plane, and the amine nitrogen of the chelate ligand and the phosphine phosphorus occupy the apical positions. These structures strictly resemble those



Figure 3. Molecular structure of Re2.

Figure 6. Molecular structure of Re6. Scheme 2. Carrier-Free Preparation of Complexes

Re ‘4 + 1’

188



of rhenium complexes and its second-row congener technetium, in which the monodentate phosphine ligand has been replaced by various isocyanide groups (8-10). Angles and bond distances comprising the [Re(NS3)] fragment, as well as the linearity of the N-Re-P spin, compare well with those reported in technetium complexes and do not deserve further comment. The Re-P bond distance elongates going from phosphite (Re6, Re-P ) 2.2228(13) Å) to phosphine complexes (Re3, Re-P ) 2.291(4); Re2, Re-P ) 2.297(2); Re4, Re-P ) 2.301(4) Å). For Re2, a hydrogen bond between one of the hydroxyl groups and the nitrogen in the backbone of the monodentate P(III) ligand is observed (see Figure 3). This additional contact may further diminish the steric hindrance of this ligand (vide infra). For Re3, both ∆ and Λ conformers were found. For clarity reasons, only the latter is displayed in Figure 4. The cyclovoltammetric profile of each complex recorded in CH2Cl2 shows only a reversible one-electron process in the anodic region which can be assigned, by coulom-

etric experiments, to the formation of Re(IV), presumably accessing the 5d3 system. The oxidation process occurs in the region from -0.05 to +0.19 V for phosphinecontaining complexes. The unique phosphite complex Re6 is more difficult to oxidize by approximately 200400 mV than similar phosphine complexes, corroborating the view that phosphite-containing compounds could be more stable toward reoxidation to perrhenate. Rhenium-188 Chemistry and Stability Studies. Preparation of the 188Re Complexes. A two-step procedure was used for the synthesis of ‘4 + 1’ 188Re complexes. At first a 188Re(III)-EDTA intermediate complex was formed by reduction of 188ReO4- with 1 mg of SnCl2 in acidic solution (pH < 3). This labile intermediate species was converted to the ‘4 + 1’ complex in a second step by contemporary addition of the NS3/P ligands (Scheme 2). The yields were not optimized and were between 50 and 90% for phosphine complexes and 20-30% for phosphite complex Re6, as assessed by TLC. Comparison of retention times obtained by means of parallel radiometric and photometric detection of nonradioactive rhenium complexes and rhenium-188 complexes established the identity of the complexes prepared at millimolar and nanomolar concentrations. Stability Studies. In vitro stabilities were determined by incubating 2-3 MBq of rhenium-188 complexes in phosphate buffer, human serum, and rat serum at room temperature. The amount of formed 188ReO4- was determined by TLC analysis. Stabilities for Re1-Re8 are shown in Table 3 and graphically illustrated in Figure 7.


Figure 7. In vitro stabilities of

188Re

Schiller et al.

complexes Re2-Re8 (standard deviations are omitted for clarity).

Re 5, which can be easily attached to biomolecules via an amide bond, was one of the most stable compounds at low activity level. Its in vitro stability was therefore further investigated under harsher conditions using 37 MBq of the complex and incubation at 37 °C in human plasma. The following amounts of 188ReO4- were found (% 188ReO4-, means ( SD, n ) 3): 9 ( 1 (1 h); 16 ( 1 (24 h); 19 ( 2 (48 h). DISCUSSION

M(III) ‘4 + 1’ complexes (M ) Tc, Re) with NS3 and isocyano ligands synthesized so far were predominantly lipophilic compounds (9, 10). Development of more hydrophilic ‘4 + 1’ species would be an important result (i) to favor renal excretion of radioactive metabolites in order to minimize exposure of radiation-sensitive organs and (ii) to open the possibility for labeling of antibodies and peptides in aqueous media. By using phosphorus(III) compounds (tertiary phosphines and phosphites of different size and lipophilicity, see Scheme 1) instead of isocyano ligands, another class of stable Re(III) ‘4 + 1’ complexes was easily accessible. Nonradioactive rhenium complexes were synthesized either starting from perrhenate or by using a prereduced rhenium(III) thiourea precursor, whereas rhenium-188 complexes were prepared using a method recently described by Seifert and co-workers (11). Since the direct reduction of 188ReO4- with NS3 and phosphorus(III) ligands in the presence of SnCl2 led to a large amount of reduced/hydrolyzed rhenium-188 compounds, we preferred an alternative way based on the formation of a prereduced 188Re(III)-EDTA complex as a labile intermediate. Following ligand-exchange with the contemporary

addition of NS3/P(III) ligands, ‘4 + 1’ complexes were obtained with up to 90% radiochemical purity. In vitro stability of these 188Re ‘4 + 1’ agents was found to be very unpredictable, but strongly dependent on the nature of the monodentate P(III) coligand (Figure 7). For example, lipophilic Re1 was completely reoxidized to 188ReO - within 1 h. Similarly, the most hydrophilic 4 complexes Re2 bearing the hydroxymethyl phosphine and Re3 comprising the cyanoethyl phosphine were not stable as well, giving large amounts of perrhenate. Conversely, more lipophilic complexes such as Re5 and Re6 were found to produce only little amounts of 188ReO4in all investigated media. Differences between stabilities of Re3 and Re6 in human plasma, rat plasma, and phosphate buffer give rise to the assumption that the in vitro stability is influenced by both biological and chemical factors. The former can be specific enzymes responsible for the cleavage of chemical bonds and/or the free radicals scavenger capability of plasma components such as human serum albumin (15-17). With the aim of understanding the strikingly different in vitro stability of these 188Re ‘4 + 1’ compounds, we prepared the corresponding nonradioactive rhenium complexes Re1-Re8, collected a series of significant physicochemical parameters (compiled in Table 4), and tried to find out which factors may govern the production of substitution-inert or hydrolyzable species. In this connection, we evidently focused our attention on the coordinated P(III) ligand. The influence of steric and electronic properties of phosphorus(III) ligands on complex reactivity has been investigated over the past decades (see a comprehensive review article published by Tolman (18)) according to the



Figure 8. Possible attack of a reactive water-derived species (A) on Re ‘4 + 1’ complexes of varying steric properties.

essential role played by transition metal-phosphorus(III) ligand complexes in homogeneous catalysis. Even if steric and electronic effects are always synergistically related and their individual analysis is difficult to perform, we tried to examine physicochemical parameters one at a time. Considering the 31P NMR shift ongoing from noncoordinated to coordinated P(III) ligands in the ‘4 + 1’ rhenium complexes, we observed that the electron density at the P atom decreases in the case of tertiary phosphines (downfield shift) and increases in the case of phosphites (upfield shift). This electron delocalization directly influenced the E° of the ReIII/ReIV couple: for example, the phosphite complex Re6 was harder to oxidize by 200-400 mV compared to all of the phosphine complexes. In principle, this property could be associated with the high in vitro stability exhibited by phosphitecontaining Re6, even if complexes showing E° values approaching the Fc/Fc+ reference couple (ca. 0.00 mV, for instance Re5 and Re8) were quite stable in vitro as well. The analysis of the electronic spectra confirms the difference between phosphine and phosphite complexes regarding their electron-withdrawing ability, but does not provide unambiguous information to discriminate between complexes of high and low in vitro stability. The observation that complexes showing better resistance to oxidation (higher E° values) are more stable in vitro toward oxidation to perrhenate appears rather obvious and seems to be only one of the factors influencing the in vitro stability. Much more information comes out from the analysis of steric factors. As pointed out by Tolman (18), the thermodynamic stability and kinetic inertness of most transition metal complexes including phosphorus(III) ligands is dominated by steric effects. The Tolman’s cone angle θ (18, 20) is the parameter utilized to describe the steric hindrance of these ligands. θ is the interior linear angle of a cone centered 2.28 Å from the center of the P atom and the edges of the cone tangential to the outermost van der Waals radii of the ligand. If we correlate θ with the amount of 188ReO4- formed (Table 4), it can be seen that the in vitro stability of Re1-Re8 strongly depends on this parameter. In fact, only P(III) ligands with θ values comprised in the 140-145° range afforded stable, sterically tight rhenium-188 compounds. In contrast, complexes including both lipophilic or hydrophilic phosphine ligands with smaller cone angles (Re1, Re2) were thermodynamically accessible in the sense that they formed in a good yield and could be completely characterized at macroscopic level with cold rhenium from common organic solutions, but were labile

and gave rise to complete reoxidation to perrhenate in the presence of water. For Re2, it has to be considered that the hydrogen bond in the phosphine’s backbone (see Figure 3) may induce a further decrease of the cone angle supporting further its instability in vitro. Analogously, phosphite ligands P(OEt)3 (θ ) 109°) and P(OiPr)3 (θ ) 130°) taken from ref 18, which were expected to yield more stable Re ‘4 + 1’ complexes on the basis of electronic considerations, showed high instability with reoxidation to perrhenate in water solutions, either at macroscopic and carrier-free levels. These findings indicate that, despite the thermodynamic stability exhibited by this class of trigonal bipyramidal Re ‘4 + 1’ compounds, the inertness in aqueous solution appeared to be the critical factor to consider for their overall stability. As sketched in Figure 8, only complexes incorporating a tailored P(III) ligand are able to generate, along with the thiolate sulfurs of the NS3 ligand, an appropriate hydrophobic core around the rhenium. Only these complexes (i.e. Re5 or Re8) are kinetically inert in aqeous solution. Less encumbering P(III) ligands (smaller θ) produced instead complexes (i.e. Re1) amenable to attack for steric reasons, and, analogously, more hydrophilic Re ‘4 + 1’ species (i.e. Re3) with a suitable P(III) cone angle were subjected to hydrolysis for solubility reasons. Despite this speculation, the mechanism could be tentatively assigned as associative: the attack of a reactive waterderived species, formed under the influence of radiation, was followed by the release of the P(III) ligand, crumbling of the Re(NS3) moiety, and reoxidation to perrhenate. Radiation and formation of radicals should be considered for causing instability of rhenium-188 complexes in vitro. The fact that the amount of perrhenate is increasing with increasing activity concentration (see Re5 in Figure 9) supports our interpretation. On the opposite side, P(III) ligands with larger cone angles such as the lipophilic triisopropylphosphine (θ ) 160°) or the hydrophilic triphenylphosphine-3,3′,3′′trisulfonic acid (θ ∼155°) did not form Re ‘4 + 1’ compounds, indicating that overencumbered ligands cannot efficiently reach the Re(NS3) moiety. According to the above data, hydrophilicity of the whole complex, which still remained an important goal of this study, had to be introduced by means of suitable functional groups on the periphery of the molecule, either on the monodentate phosphine and/or on the chelate NS3 framework. The increase of the overall hydrophilicity in ‘4 + 1’-type compounds has already been demonstrated in analogous Tc complexes (11) by insertion of a carboxyl group into the NS3 chelate. The incorporation of a carboxyl group could also serve for the conjugation of


Schiller et al.

periphery of the complex by insertion of carboxylic functions, either at the NS3 framework and/or at the monodentate phosphine ligand. 188Re ‘4 + 1’ complexes of this formulation were found to be sufficiently stable toward reoxidation in phosphate buffer as well as in rat plasma and human plasma, both at low and high activity levels. Therefore, we consider these agents as promising candidates for the development of 188Re radiotherapeutics. ACKNOWLEDGMENT

Figure 9. Comparison of in vitro stabilities of 188Re(CO)3{[(di1H-imidazol-2-ylmethyl)amino] acetic acid} (4) and Re5 after incubation in human plasma at 37 °C.

biomolecules. Such peripheral variation does not affect the in vitro stability of the complexes. For example, Re7 and Re8, carrying a free carboxyl group or an amide group onto the NS3 backbone exhibited higher hydrophilicity (Re7 . Re8 > Re5) but comparable in vitro stability than their unsubstituted analogue Re5. Only few and mostly imprecise data on the stability of 188Re complexes are found in the literature. Complexes with mercaptoacetyl triglycine (188Re(O)MAG3) and those of the HYNIC system were found to be unstable in vitro and in vivo by our and other groups (21-23). Contrarily, some groups found high in vitro and in vivo stability of 188Re(O)MAG even at high activity concentration (53 7). Schibli and co-workers reported on the in vitro stability of 188Re-tricarbonyl complexes incorporating different tridentate ligands (4), but utilizing higher activity and higher temperature (37 MBq, 37 °C). We compared the in vitro stability of Re5 under these conditions with that displayed by the most stable tricarbonyl complex having [(di-1H-imidazol-2-ylmethyl)amino]acetic acid as tridentate ligand and found similar in vitro stabilities (Figure 9). CONCLUSION

A series of Re(III) ‘4 + 1’ complexes, [ReNS3)(PRR′R′′)], containing a tetradentate NS3 chelator and a monodentate phosphorus(III) coligand (tertiary phosphine or phosphite) has been prepared both at macroscopic level with nonradioactive rhenium and at carrier-free level with 188Re. In vitro stabilities of 188Re ‘4 + 1’ complexes in human plasma, rat plasma, and in phosphate buffer depend primarily on the nature of the phosphorus(III) coligand. Several physicochemical parameters of homologous rhenium complexes including ReIII/ReIV redox potentials and electronic and NMR spectra, together with scrutiny of the steric hindrance of P(III) ligands, were consequently analyzed in order to rationalize the in vitro behavior. The Tolman’s cone angle θ was found to be the crucial factor in the formation of substitution-inert Re(III) ‘4 + 1’ complexes. Only tertiary phosphines and phosphites with cone angles in the range between 140 and 145° gave rise to agents stable in vitro. Obviously, the steric hindrance of these P(III) coligands along with the lipophilic nature of the three coordinated thiolate sulfurs generates a hydrophobic area around the Re(III) center which prevents hydrolysis and reoxidation to perrhenate. Hence, the initial idea to synthesize overall hydrophilic Re ‘4 + 1’ compounds was changed, and, while preserving a lipophilic core in the inner coordination sphere, hydrophilicity was introduced into the

We thank G. Wunderlich (Klinik und Poliklinik fu¨r Nuklearmedizin, TU Dresden) for providing a 188W/188Re generator and the Deutscher Akademischer Austauschdienst (DAAD) for financial support. LITERATURE CITED (1) Knapp, F. F. (1998) Rhenium-188 - A generator-derived radioisotope for cancer therapy. Cancer Biother. Radiopharm. 13, 337-349. (2) Knapp, F. F., Beets, A. L., Guhlke, S., Zamora, P. O., Bender, H., Palmedo, H., and Biersack, H.-J. (1997) Availability of rhenium-188 from the alumina-based tungsten-188/rhenium188 generator for preparation of rhenium-188-labeled radiopharmaceuticals for cancer treatment. Anticancer Res. 17, 1783-1796. (3) Jeong, J. M., and Chung, J.-K. (2003) Therapy with 188Relabeled radiopharmaceuticals: An overview of promising results from initial clinical trials. Cancer Biother. Radiopharm. 5, 707-717. (4) Schibli, R., Schwarzbach, R., Alberto, R., Ortner, K., Schmalle, H., Dumas, C., Egli, A., and Schubiger, P. A. (2002) 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. Bioconjugate Chem. 13, 750-756. (5) Lin, W. Y., Tsai, S. C., Hsieh, B. T., Lee, T. W., Ting, G., and Wang, S. J. (2000) Evaluation of three rhenium-188 candidates for intravascular radiation therapy with liquidfilled balloons to prevent restenosis. J. Nucl. Cardiol. 7, 3742. (6) Guhlke, S., Schaffland, A., Zamora, P. O., Sartor, J., Diekman, D., Bender, H., Knapp, F. F., and Biersack, H.-J. (1998) 188Re- and 99mTc-MAG3 as prosthetic groups for labeling amines and peptides: Approaches with pre- and postconjugate labeling. Nucl. Med. Biol. 25, 621-631. (7) Oh, S. J., Moon, D. H., Ha, H. J., Park, S. W., Hong, M. K., Park, S. J., Choi, T. H., Lim, S. M., Choi, C. W., Knapp, F. F., and Lee, H. K. (2001) Automation of the synthesis of highly concentrated 188Re-MAG3 for intracoronary radiation therapy. Appl. Radiat. Isot. 54, 419-427. (8) Spies, H., Glaser, M., Pietzsch, H.-J., Hahn, F. E., and Lu¨gger, T. (1995) Synthesis and reactions of trigonal-bipyramidal rhenium and technetium complexes with a tripodal, tetradentate NS3 ligand. Inorg. Chim. Acta 240, 465-478. (9) Drews, A., Pietzsch, H.-J., Syhre, R., Seifert, S., Varna¨s, K., Hall, H., Halldin, C., Kraus, W., Karlsson, P., Johnsson, C., Spies, H., and Johannsen, B. (2002) Synthesis and biological evaluation of technetium(III) mixed-ligand complexes with high affinity for the cerebral 5-HT1a receptor and the alpha1adreneric receptor. Nucl. Med. Biol. 29, 389-398. (10) Pietzsch, H.-J., Gupta, A., Syhre, R., Leibnitz, P., and Spies, H. (2001) Mixed-ligand technetium(III) complexes with tetradentate/monodentate NS3/isocyanide coordination: A new nonpolar technetium chelate system for the design of neutral and lipophilic complexes stable in vivo. Bioconjugate Chem. 12, 583-544. (11) Seifert, S., Ku¨nstler, J.-U., Schiller, E., Pietzsch, H.-J., Pawelke, B., Bergmann, R., and Spies, H. (2004) Novel procedures for preparing 99mTc complexes with tetradentate/ monodentate coordination of varying lipophilicity and adaption to 188Re analogues. Bioconjugate Chem. 15, 856-863.


Mixed-Ligand Rhenium-188 Complexes (12) Jarolim, T., and Podlahova, J. (1976) Coordinating behaviour of diphenylphosphineacetic acid. J. Inorg. Nucl. Chem. 38, 125-129. (13) Kosolapoff, G. M. (1948) Isomerization of trialkyl phosphites. Some derivatives of 2-bromoethane phosphonic acid. J. Am. Chem. Soc. 70, 1971-1972. (14) Gambino, D., Otero, L., Kremer, E., Piro, O. E., and Castellano, E. E. (1997) Synthesis, characterization and crystal structure of hexakis-(thiourea-S)-rhenium(III) trichloride tetrahydrate: A potential precursor to low-valent rhenium complexes. Polyhedron 16, 2263-2270. (15) Chakrabarti, M. C., Le, N., Paik, C. H., de Graff, W. G., and Carrasquillo, J. A. (1996) Preventions of radiolysis of monoclonal antibody during labeling. J. Nucl. Med. 37, 13841388. (16) Salako, Q. A., O’Donell, R. T., and DeNardo, S. J. (1998) Effects of radiolysis on yttrium-90-labeled Lym-1 antibody preparations. J. Nucl. Med. 39, 667-670. (17) Iznaga-Escobar, N. (2001) Direct radiolabeling of monoclonal antibodies with rhenium-188 for radioimmunotheraphy of solid tumors - A review of radiolabeling characteristics, quality control and in vitro stability studies. Appl. Radiat. Isot. 54, 399-406.

(18) Tolman, C. A. (1977) Steric effects of phosphorus ligands in organometallic chemistry and homogeneous catalysis. Chem. Rev. 77, 313-348. (19) Rahman, M., Liu, H. Y., Prock, A., and Giering, W. (1987) Steric and electronic factors influencing transition-metalphosphorus(III) bonding. Organometallics 6, 650-658. (20) Tolman, C. A. (1970) Phosphorus ligand exchange equilibria on zerovalent nickel. A dominant role for steric effects. J. Am. Chem. Soc. 92, 2956-2965. (21) He, J., Liu, C., Venderheyden, J. L., Liu, G., Dou, S., Ruschkowski, M., and Hnatowich, D. J. (2003) Radiolabeling morpholinos with 188Re carbonyl provides improved in vitro and in vivo stability to re-oxidation. J. Nucl. Med. 44, 100P. (22) Liu, C.-B., Liu, G.-B., Liu, N., Zhang, Y.-M., He, J., Rusckowski, M., and Hnatowich, D. J. (2003) Radiolabeling morpholinos with 90Y, 111In, 188Re, 99mTc. Nucl. Med. Biol. 30, 207-214. (23) Seifert, S., Ku¨nstler, J.-U., and Johannsen, B. (2002) Stability of 99mTc and 188Re labelled HYNIC-IVIG. Wissenschaftlich-Technische Berichte FZR-340, Annual Report 2001; p 42.

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Mixed-Ligand Rhenium-188 Complexes with Tetradentate

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