Dramatic Effect of the Tridentate Ligand on the Stability of 99mTc "3 +

Bioconjugate Chem. 2005, 16, 660−668

660

Dramatic Effect of the Tridentate Ligand on the Stability of "3 + 1" Oxo Complexes Bearing Arylpiperazine Derivatives

99mTc

C. Fernandes,† J. D. G. Correia,† L. Gano,† I. Santos,*,† S. Seifert,‡ R. Syhre,‡ R. Bergmann,‡ and H. Spies‡ Departamento de Quı´mica, ITN, Estrada Nacional 10, 2686-953, Sacave´m Codex, Portugal, and Institute of Bioinorganic & Radiopharmaceutical Chemistry, Forschungszentrum Rossendorf, D-01314 Dresden, Germany . Received November 22, 2004; Revised Manuscript Received March 28, 2005

Mixed - ligand model complexes of general formula [99mTc(O)(κ3-PNX)(κ1-SPh))] [X ) O (1a), S (2a)] were prepared in a one-step procedure from [99mTcO4-] using stannous chloride as reducing agent. Stability studies and challenge experiments with glutathione showed that complex 2a presented promising features for pursuing animal studies. The activity in the brain (% dose injected/organ) at 5 min (0.14% ( 0.03) and 120 min (0.11% ( 0.02) pi encouraged the synthesis of several mixed-ligand "3 + 1" oxo complexes of general formula [M(O)(κ3-PNS)(κ1-SL))] (M ) 99mTc, 3a-6a, Re, 3-6), in which the tridentate ligand is the heterofunctionalized phosphine 2-(diphenylphosphanyl)-N-(2thioethyl)benzamide (PNS) and the co-ligands are different arylpiperazine derivatives (HSL1-HSL4). The 99mTc complexes have been characterized by comparison of their retention times in the HPLC chromatogram (γ-detection) with the retention times of the analogous Re complexes (UV detection at 254 nm). The 99mTc complexes, obtained with radiochemical purity higher than 95%, after HPLC purification, are stable in saline, 0.01 M PBS (pH 7.4), rat plasma (4 h, 37 °C), and glutathione (10 mM solutions, 2h, 37 °C). Binding affinity and selectivity for 5-HT1A receptors (relative to the 5-HT2A receptor) were determined, complex 5 demonstrating the best values (IC50 for the 5-HT1A 2.35 ( 0.02 nM; competitor 5-HT2A 372 ( 11 nM). Biodistribution and stability studies in mice indicated a preferred hepatobiliary excretion, a high in vivo stability, but a poor brain uptake.

INTRODUCTION

Scheme 1

One of the major challenges in the development of new specific radiopharmaceuticals based on 99mTc, the radionuclide of choice in nuclear medicine, is to bind a biomolecule to the metal center without drastically modifying its biological activity and metabolism. The development of 99mTc-based radiopharmaceuticals for studying the receptor function in the central nervous system (CNS) requires neutral and lipophilic complexes to facilitate their penetration through the blood-brain barrier (BBB), maintaining the affinity and selectivity for the neuroreceptor. Despite the difficulties, several approaches have been investigated to achieve that purpose (1-3). The successful development of [99mTc]TRODAT-1 as a 99mTc-radiopharmaceutical for imaging the dopamine transporters in the human brain was a very encouraging result (4, 5). The so-called "3 + 1" approach has been thoroughly investigated in the past few years for the design and preparation of specific radiopharmaceuticals of rhenium and technetium (6-10). This method uses tridentate dithiolate ligands HSESH (E ) S; NR; O) and monodentate thiolate (HSR) co-ligands to stabilize the core [Tc(O)]3+ and to form neutral and lipophilic complexes of general formula [Tc(O)(SES)(SR)]. However, one of the problems associated with the use of such systems is the low stability of these complexes * To whom correspondence should be addressed. E-mail: [email protected]. † Estrada Nacional 10. ‡ Institute of Bioinorganic & Radiopharmaceutical Chemistry.

against thiolated nucleophiles such as glutathione. In fact, although stable in PBS solutions, they presented low stability in vivo. Challenge experiments against glutathione (GSH) showed that their in vivo instability is mediated by the nucleophilic substitution of the monodentate co-ligand by glutathione with formation of the hydrophilic complex [99mTc(O)(SES)(SG)] (E ) S, NR). Such instability was considered to be strongly dependent on the nature of the tridentate/monodentate ligands but, so far, no tri/monodentate pair has been found to overcome this problem (11-13). The (tri)bidentate heterofunctionalized phosphines, 2-(diphenylphosphanyl)-N-(2-hydroxyethyl)benzamide (H2PNO) and 2-(diphenylphosphanyl)-N-(2-thioethyl)benzamide (H2PNS) (Scheme 1), which are quite versatile in terms of charge and denticity toward the [MdO]3+ core, form "3 + 1" mixed-ligand oxo complexes of general formula [Re(O)(PNX)(SR)] (X ) O, S) with different thiolated monodentate co-ligands, some of them presenting a high stability (14-16). Visualization of brain serotonin receptors, particularly the 5-HT1A subtype, is of great interest since these receptors are implicated in various neuropsychiatric diseases (17, 18). The aim of our work is the preparation of novel mixed-ligand 99mTc oxo complexes with potential for in vivo assessment of 5-HT1A receptors.

10.1021/bc049718k CCC: $30.25 © 2005 American Chemical Society Published on Web 04/29/2005

99mTc

"3 + 1" Oxo Complexes

Herein we report on the synthesis, characterization, and biological evaluation of mixed-ligand model complexes of general formula [99mTc(O)(κ3-PNX)(κ1-SPh))] [X ) O (1a), S (2a)]. The complexes [M(O)(κ3-PNS)(κ1-SL)] (M ) 99mTc, 3a-6a, Re, 3-6), anchored by the H2PNS ligand and by several thiolated arylpiperazine derivatives (HSL1-HSL4) will also be described (Scheme 4). The binding affinity and specificity of 3-6 for the 5HT1A receptors, the in vitro and in vivo stability, and the biokinetics of 3a-6a are also reported. EXPERIMENTAL SECTION

Materials and Methods. Unless otherwise stated, all chemicals were of reagent grade and were used without further purification. The heterofunctionalized phosphines, 2-(diphenylphosphanyl)-N-(2-hydroxyethyl)benzamide (H2PNO), 2-(diphenylphosphanyl)-N-(2-thioethyl)benzamide (H2PNS), and succinimido 3-[(triphenylmethyl)thio]propionate, were prepared as previously reported (14, 15, 19). The monothiols, thiophenol, p-aminothiophenol, mercaptopropionic acid, benzylmercaptane, cyclohexanethiol, and L-glutathione (GSH), were commercially available. [nBu4N][Re(O)Cl4], [Re(O)Cl3(PPh3)] and [Re(O)(κ3-PNX)(κ1-SPh))] (X ) O (1), ) S (2)) were prepared according to literature methods (20, 21, 14, 15). Sodium pertechnetate was obtained in saline solution from a commercial 99Mo/99mTc generator (MDS Nordion S.A., Belgium or Mallinckrodt). Elemental analysis was performed on a Perkin-Elmer automatic analyzer. Infrared spectra were recorded in the range 4000-200 cm-1 on a Perkin-Elmer 577 spectrometer from KBr pellets. The1H and 31P NMR spectra were recorded on a Varian Unity 300 MHz spectrometer; 1H chemical shifts were referenced relative to tetramethylsilane and the 31P chemical shifts to external 85% H3PO4 solution. The NMR samples were prepared in CDCl3, chemical shifts are given in ppm. Mass spectrometric analyses were carried out on a Micromass Tandem Quadrupole Mass Spectrometer (Quattro LC) operating in the MS mode. Mass spectral data were recorded at the negative and positive ESI mode using a cone voltage of 20 or 60 V. About 0.1 mg of the sample dissolved in 1.0 mL methanol/water (50/50), and 0.05% formic acid was infused at a flow rate of 5 µL/min. Chemical reactions were monitored by thin-layer chromatography (TLC) on Merck plates precoated with silica gel 60 F254. Spots were visualized either by UV light and/ or Ellman’s reagent for thiols. Column chromatography was performed in silica gel 60 (Merck). High performance liquid chromatography (HPLC) analysis was performed on a Perkin-Elmer system coupled with two detectors: a UV-Vis detector, LC 290, Perkin-Elmer (UV detection for ligands and Re complexes at 254 nm) and a γ-detector (LB 509, Berthold) for 99mTc compounds. Separations were achieved on a reversed phase (RP) Hypersil ODS column (4.0 × 250 mm, 10 µm) eluted with a binary gradient system with a flow rate of 1.0 mL/min. Mobile phase A is methanol while mobile phase B is 0.01 M phosphate buffer (pH 7.4). The elution profile used was a linear gradient starting with 75% A/25% B to 95% A/5% B in 10 min, and this composition was held for 5 min. The 99mTc model complexes were analyzed by TLC using two different chromatographic systems: (a) Silufol/(BuOH/ MeOH/water/ammonia) (60/20/20/1); (b) Silufol/saline. Synthesis of Monodentate Thiolate Ligands Bearing Arylpiperazine Derivatives. 3-Mercapto-1-[4-(2methoxyphenyl)-piperazin-1-yl]propan-1-one (HSL2). Ph3CSCH2CH2C(O)PIP (where PIP is 4-(2-methoxyphenyl)-

Bioconjugate Chem., Vol. 16, No. 3, 2005 661

piperazin-1-yl): 1-(2-Methoxyphenyl)piperazine (0.29 g, 1.5 mmol) was dissolved in 5 mL of dichloromethane and stirred at room temperature (r.t.) under a nitrogen atmosphere. A solution of succinimido 3-[(triphenylmethyl)thio]propionate (0.67 g, 1.5 mmol) dissolved in the same solvent (10 mL) and triethylamine (110 µL, 1.5 mmol) was added dropwise. After 22 h the reaction was completed, a diluted solution of HCl was added, and the reaction mixture was extracted three times with dichloromethane. The organic phase was collected, washed with water, dried over MgSO4, filtered, and vacuum-dried. A viscous oil, which afforded a white solid after being vacuum-dried, was obtained (0.70 g, 90%). IR (cm-1, KBr): 1635 (CdO), 1490, 1240, 1200, 750, 700. 1H NMR (δ, ppm, CDCl3): 2.14 (t, 2H, CH2), 2.56 (t, 2H, CH2), 2.94 (br s, 4H, CH2), 3.34 (br s, 2H, CH2), 3.70 (br s, 2H, CH2), 3.85 (s, 3H, OCH3), 7.03-6.85 (m, 4H, CHar), 7.42-7.14 (m, 15H, CHar). Elemental analysis: C33H34O2N2S calcd, C 75.83, H 6.56, N 5.36. found, C 75.14, H 8.30, N 4.96. Ph3CSCH2CH2C(O)PIP (0.30 g, 0.58 mmol) was dissolved in 10 mL of trifluoroacetic acid (TFA) and cooled in an ice bath, and triethylsilane (0.2 mL 1.2 mmol) was added. The color of the initial solution (yellow) discharged, and a white precipitate was formed. After stirring for 15 min more, the solution was diluted with a mixture of hexane/water. The aqueous phase was separated, washed three times with hexane, and extracted three times with dichloromethane (25 mL). The organic phase was collected, washed with a saturated solution of sodium bicarbonate and water, and finally dried over MgSO4. After filtration, the solvent was evaporated and the colorless oil obtained became a white solid on standing. This compound was formulated as HSL2 based on the analytical data (0.12 g, 74%). IR (cm-1, KBr): 1635 (CdO), 1490, 1240, 1200, 750, 700. 1H NMR (δ, ppm, CDCl3): 1.75 (t, 1H, SH), 2.68 (t, 2H, CH2), 2.82 (q, 2H, CH2), 3.05-2.99 (m, 4H, CH2), 3.63 (t, 2H, CH2), 3.79 (t, 2H, CH2), 3.85 (s, 3H, OCH3), 7.03-6.86 (m, 4H, CHar). Elemental analysis: C14H20O2N2S calcd, C 59.99, H 7.19, N 9.99 S 11.43, found, C 58.99, H 6.80, N 9.92 S 11.04. Synthesis of 3-[4-(2-Methoxyphenyl)piperazin-1-yl]propane-1-thiol (HSL3). To a solution of Ph3CSCH2CH2C(O)PIP (346 mg, 0.66 mmol) in dichloromethane (10 mL) was added dropwise 1 M borane-methyl sulfide solution (2.6 mL, 2.6 mmol) and the mixture stirred for 18 h at room temperature, under nitrogen atmosphere. The reaction mixture was cooled and methanol added dropwise until gas formation was ceased. The solvent was evaporated under vacuum, dichloromethane was added, and the mixture was refluxed for 15 min. After removal of the solvent, the crude product obtained was purified by column chromatography (silica gel), using as eluent dichloromethane/methanol (9:1). Ph3CSCH2CH2CPIP was obtained as a white solid, after removing the solvent under vacuum (0.24 g, 72%). After deprotection, as abovedescribed for HSL2, compound HSL3 was obtained as a white solid (0.12 g, 74%). IR (cm-1, KBr): 1490, 1240, 1200, 750, 700. 1H NMR (δ, ppm, CDCl3): 1.40 (t, 1H, SH), 1.83 (q, 2H, CH2), 2.51 (t, 2H, CH2), 2.57 (t, 2H, CH2), 2.64 (br s, 4H, CH2), 3.01 (br s, 4H, CH2), 3.84 (s, 3H, OCH3), 6.95-6.82 (m, 4H, CHar). Elemental analysis: C14H22ON2S calcd, C 63.12, H 8.32, N 10.52, S 12.03, found, C 62.99, H 9.10, N 9.92 S 11.04. Synthesis of 4-Mercapto-N-{3-[4-(2-methoxyphenyl)piperazin-1-yl]propyl}benzamide (HSL4). Triphenylmethylmercaptobenzoic Acid: Triphenylmethanol (1,04 g, 4 mmol) was added to a suspension of mercaptobenzoic acid (0.62 g, 4 mmol) in TFA yielding a yellow solution. After 1 h at room temperature, under stirring, the solution was

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

evaporated to dryness. The resulting yellow solid was dissolved with ethyl acetate and washed with 3 N NaOH solution, water, a saturated solution of sodium bicarbonate, and finally with brine. The organic phase was dried over MgSO4, filtered, and after vacuum-drying, triphenylmethylmercaptobenzoic acid was obtained as a solid (1.54 g, 97%). The compound was used without any further purification. Triphenylmethyl(mercaptobenzoyloxy)succinimide: Triphenylmethylmercaptobenzoic acid (1.41 g, 3.55 mmol) and N-hydroxysuccinimide (0.87 g, 7.4 mmol) were dissolved in dichloromethane (20 mL), and dicyclohexylcarbodiimide (1.54 g, 7.4 mmol) dissolved in the same solvent was added dropwise. After 4 h the reaction was complet (TLC/ethyl acetate/hexane (1:1)) and the urea formed was removed by filtration through Celite. The solution was vacuum-dried and the solid obtained recrystallized from ethyl acetate. The white solid obtained was formulated as triphenylmethyl(mercaptobenzoyloxy)succinimide, based on analytical data (1.48 g (85%). 1H NMR (δ, ppm, CDCl3): 2.85 (s, 4H, CH2), 7.00 (d, J ) 8.1 Hz, 2H, CHar), 7.24-7.38 (m, 15H, CHar), 7.70 (d, J ) 8.1 Hz, 2H, CHar). Elemental analysis: C30H23O4NS calcd, C 73.02, H 4.66, N 2.83 S 6.49, found, C 72.21, H 5.47, N 2.24, S 7.07. Triphenylmethyl(mercaptobenzoyloxy)succinimide (0.25 g, 0.5 mmol), 4-(3-aminopropyl)-1-(methoxyphenyl)piperazine (0.13 g, 0.5 mmol), and triethylamine (50 µL) reacted in dichloromethane (30 mL) at room temperature, under nitrogen atmosphere. After 18 h, the reaction was complete. A diluted solution of HCl was added and the aqueous phase extracted three times with dichloromethane. The organic phase was collected, washed with water, and dried over MgSO4. After evaporation of the solvent a white solid was obtained, which after deprotection of the thiol group was formulated as HSL4 (0.11 g, 75%). IR (cm-1, KBr): 1630 (CdO), 1590, 1240, 740, 695. 1H NMR (δ, ppm, CDCl3): 2.00 br s, 2H, CH2), 2.85 (br s, 2H, CH2), 2.99 (br s, 4H, CH2), 3.26 (br s, 4H, CH2),), 3.60 (br s, 2H, CH2), 3.84 (s, 3H, OCH3), 7.85-6.86 (m, 8H, CHar), 8.41 (t, 1H, NH). Elemental analysis: C21H27O2N3S calcd, C 65.45, H 7.01, N 10.91 S 8.31, found, C 64.95, H 8.02, N 10.77, S 9.16. General Procedure for the Preparation of Re Complexes. To a stirred suspension of [Re(O)Cl3(PPh3)2] in a 0.15 M methanolic solution of sodium acetate (10 mL) was added a mixture of H2PNS with the corresponding thiolated aryl piperazine derivative in the same solution (5 mL). The suspension was stirred and refluxed for 1 h, yielding a dark brown solution. After cooling to room temperature, the reaction mixture was diluted with dichloromethane and with a 0.01 M HCl solution. The aqueous phase was then extracted three times with dichloromethane, and the organic phases were collected. After drying over magnesium sulfate, the solution was filtered and the solvent evaporated. The dark brown residue obtained was washed three times with diethyl ether and purified by chromatography using a silica gel column and a mixture of methanol-chloroform as eluent. [Re(O)(PNS)(SL2)] (4). IR (cm-1, KBr): 1630 (CdO), 1600 (CdO), 970 (RedO), 745, 690. 1H NMR (δ, ppm, CDCl3): 8.13 (m, 1H, CHar), 7.67-7.18 (m, 12H, CHar), 7.03-6.85 (m, 4H, CHar), 6.79-6.73 (m, 1H, CHar), 5.05 (m, 1H, CH of tridentate ligand), 4.15 (m, 2H, SCH2), 3.84 (s, 3H, OCH3), 3.73 (br s, 4H, CH2 of piperazine), 3.012.92 (m, 2H, CH2 of piperazine, CH2 of monodentate ligand, CH of tridentate ligand), 2.78-2.69 (m, 1H, CH of tridentate ligand), 2.19 (m, 1H, CH of tridentate ligand); 31P NMR (δ, ppm, CDCl3): 20.0. Elemental

Fernandes et al.

analysis: C35H37O4N3PS2Re calcd, C 49.75, H 4.41, N 4.97 S 7.59, found, C, H, N, S. [Re(O)(PNS)(SL3)] (5). IR (cm-1, KBr): 1590 (CdO), 970 (RedO), 750, 690. 1H NMR (δ, ppm, CDCl3): 8.158.11 (m, 1H, CHar), 7.67-7.22 (m, 12H, CHar), 6.92-6.71 (m, 5H, CHar), 5.06 (m, 1H, CH of tridentate ligand), 3.83 (s, 3H, OCH3), 3.59 (m, 2H, CH2 of propyl), 3.07 (br s, 4H, CH2 of piperazine), 2.97 (2.73-2.61 (m, 6H, CH2 of piperazine, CH2 of propyl), 2.60-2.55 (m, 1H, CH of tridentate ligand), 2.20 (m, 1H, CH of tridentate ligand), 1.83 (m, 2H, CH2 of propyl); 31P NMR (δ, ppm, CDCl3): 19.9. Elemental analysis: C35H39O3N3PS2Re calcd, C 50.59, H 4.73, N 5.06, S 7.72, found, C, H, N, S. [Re(O)(PNS)(SL4)] (6). IR (cm-1, KBr): 1640 (CdO), 1590 (CdO), 975 (RedO), 750, 700. 1H NMR (δ, ppm, CDCl3): 8.37 (t, 1H, NH), 8.17-8.13 (m, 1H, CHar), 7.877.24 (m, 16H, CHar), 6.92-6.71 (m, 5H, CHar), 5.05 (m, 1H, CH of tridentate ligand), 3.83 (s, 3H, OCH3), 3.59 (m, 2H, CH2 of propyl), 3.09 (br s, 4H, CH2 of piperazine), 2.73-2.61 (m, 6H, CH2 of piperazine, CH2 of propyl), 2.60-2.55 (m, 1H, CH of tridentate ligand), 2.20 (m, 1H, CH of tridentate ligand), 1.83 (m, 2H, CH2 of propyl); 31P NMR (δ, ppm, CDCl3): 19.6. Elemental analysis: C42H44O4N4PS2Re calcd, C 53.09, H 4.60, N 5.90 S 6.75, found, C, H, N, S. 99m Tc Complexes. [99mTc(O)(PNO)(SPh)] (1a). The preparation of the complexes at the tracer level was accomplished in a one step procedure, starting with [99mTcO4-] and using stannous chloride as reducing agent. The following general procedure was used: A mixture of propylene glycol (50 µL), ethanol or acetonitrile (1300 µL), 0.5 mg (4.5 × 10-6 mol) of thiophenol, 0.05 mg (1.43 × 10-7 mol) of H2PNO, 0.1 N NaOH (200 µL), 20 µL of a SnCl2 solution (1.0-2.0 mg SnCl2 dissolved in 5 mL 0.1 N HCl), and 0.5 mL of [99mTc]pertechnetate eluate (100200 MBq) was heated at 37 °C over 20 min. The reaction mixture was analyzed by HPLC, using the conditions described above and also by TLC using two different chromatographic systems: (a) Silufol/(BuOH/MeOH/ water/ammonia) (60/20/20/1); (b) Silufol/saline. Yields > 90% were obtained. The complexes were purified by HPLC on a semipreparative Hypersil ODS column (Hypersil 120 ODS, 250 × 8 mm, 10 µm, flow rate 2.0 mL/min) using a linear gradient (t[min]/%A: (5/75), (10/95), (10/95) of methanol (A)/0.01 M phosphate buffer (pH 7.4) (B). Characterization of the 99mTc complexes was accomplished by chromatographic correlation (HPLC) with the corresponding rhenium complexes, previously described (14, 15). [99mTc(O)(PNS)(SPh)] (2a). In the case of the H2PNS ligand, the "3 + 1" mixed-ligand complex is not formed under alkaline conditions. So, the same procedure described above was used but in the absence of NaOH. Compound [99mTc(PNS)(SPh)] was obtained in relatively good yield (>90%). [99/99mTcO(PNS)(SPh)]. Complex [99/99mTcO(PNS)(SPh)] was prepared as described for 2a, using a [99mTcO4-] eluate enriched with 99Tc (10-8 or 10-7 mol 99TcO4-). The characterization was done by HPLC using the Re complex as a surrogate and also by mass spectrometry. General Procedure for the Preparation of 99mTc Complexes (3a-6a). The 99mTc complexes were prepared by direct reduction of pertechnetate with stannous chloride. The following general procedure was used: 50 µL of propylene glycol (PG), 1300 µL of ethanol, 0.030.05 mg of the monodentate ligand, 0.001 mg (1.4 × 10-6 mol) of H2PNS, and 20 µL of a SnCl2 solution (2.0-3.0 mg of SnCl2 dissolved in 5 mL of 0.1 N HCl) were added

99mTc


to 0.5 mL (100-200 MBq) of [99mTc]pertechnetate eluate. The reaction mixture was heated at 40-50 °C for 20 min. The 99mTc complexes were analyzed by HPLC using a reversed-phase Hypersil 120 ODS column (250 × 4 mm, 10 µm). The column was eluted using methanol/water as mobile phase at 1.0 mL/min flow rate and with a linear gradient of 70-100% methanol over 10 min followed by 10 min at 100% of methanol. These complexes were also analyzed by TLC using two different chromatographic systems: (a) silica gel/acetone; (b) silica gel/saline. The compounds were purified on a semipreparative Hypersil ODS column (250 × 8 mm, 10 µm) using methanol/water as mobile phase (flow rate: 2 mL/min) and with a linear gradient of 70-100% methanol over 10 min followed by 10 min at 100% of methanol. The fractions were collected and analyzed in the analytical column. The purified complexes, after removal of the organic solvent, were reconstituted with 0.1 M PBS 7.4, and 100 µL of PG was added. The characterization of the 99m Tc complexes was accomplished by chromatographic correlation (HPLC) with the corresponding rhenium complexes. General Procedures for Stability Studies in Vitro. Challenge with GSH. 100 µL (approximately 10 MBq) of the purified 99mTc complex was mixed with 100 µL of a 2 mM or 20 mM solution of GSH in 0.1 M phosphate buffer (pH 7.4). The mixture was incubated at 37 °C for 5-120 min and analyzed by HPLC. Samples without GSH were also analyzed and used for comparison. HPLC analyses were carried out using the experimental conditions above-described. Stability in Rat Plasma. Blood collected from mice in heparinized polypropylene tubes was immediately centrifuged for 15 min at 2000 rpm at 4 °C and the plasma collected. The 99mTc complex (100 µL, ≈10 MBq) was added to fresh rat plasma (1 mL), and the mixture was incubated at 37 °C. At appropriate periods of time (5 min, 30 min, 1, 2, and 4 h), 100 µL aliquots (in duplicate) were sampled and treated with 200 µL of ethanol to precipitate the proteins. Samples were then cooled at 4 °C and centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant was separated from the precipitate, and the sediment was washed twice with ethanol (1 mL) and counted in a gamma counter. The activity in the sediment was compared with the total activity used, and the percent of complex bound to proteins was calculated. The supernatant was analyzed by HPLC, using the experimental conditions above-described. Stability in Whole Human Blood. To whole human blood (1 mL), collected in heparinized polypropylene tubes, was added a solution of the 99mTc-complex (≈10 MBq), and the mixture was incubated at 37 °C. Samples were taken 5 min, 1 h, and 4 h after incubation and centrifuged 15 min at 2000 rpm at 4 °C. The plasma was separated and ethanol was added in a 2:1 (v/v) ratio. The samples were centrifuged at 3000 rpm, and the supernatant was analyzed by HPLC. Receptor Binding Assays on Rat Brain Homogenates. The 5HT1A receptor binding assays were performed using [3H]-8-OH-DPAT as radioligand and rat hippocampus homogenate. The binding assay was carried out in a final volume of 2.5 mL of Tris-HCl buffer (50 mM, pH 7.4, 0.1% ascorbic acid, 2 mM CaCl2) containing 0.10 nM [3H]-8-OH-DPAT, membrane homogenate (about 20 µg/mL protein), and various concentration of the rhenium complexes 3-6. The complexes were dissolved in DMSO up to 1 nM and then diluted with buffer. Nonspecific binding was defined as the amount of [3H]8-OH-DPAT bound in the presence of serotonin (Sigma).


The samples were incubated in triplicates at 20 °C for 120 min. The incubation was terminated by rapid filtration through GF/B glass fiber filters (Whatman) using a 30-port Brandel Cell Harvester. The filters were rapidly washed with four portions of ice-cold buffer, transferred into 10 mL of scintillation fluid (Ultima-Gold, Packard), and analyzed for radioactivity. The 5HT2A receptor binding assays: The cortex of rat brain was homogenized in ice-cold buffer (50 mM trisHCl, pH 7.6) with an Ultra-Turrax T 25. The homogenate was centrifuged at 20 000g for 10 min. The resulting pellet was resuspended and centrifuged under the above conditions. After repeating the same procedure, the pellet was resuspended in 10 mL of buffer and stored at -20 °C. [3H]Ketanserin (from NEN) was used as a radioligand. The binding assay was carried out in a final volume of 5 mL of Tris-HCl buffer, pH 7.6, containing 0.12 nM [3H]ketanserin, membrane homogenate (about 20 µg/mL protein), and various concentrations of the Re complexes. The complexes were dissolved in DMSO up to 1 nM and then diluted with buffer. Nonspecific binding was defined as the amount of [3H]ketanserin bound in the presence of 1 µM mianserin (Sigma). The samples were incubated in triplicates at 20 °C for 60 min. Incubation, filtration, and counting of the samples were the same as described above. General Procedures for in Vivo Studies. Biodistribution Studies. The in vivo behavior of the 99mTc complexes was evaluated in groups of 3-5 female CD-1 mice (randomly bred, Charles River) weighing approximately 20-25 g each. Animals were intravenously injected with 100 µL (1-3 MBq) of each preparation via the tail vein and were maintained on normal diet ad libitum. At 5 min and 1 h mice were killed by cervical dislocation. The radioactive dosage administered and the radioactivity in the sacrificed animal were measured in a dose calibrator (Aloka, Curiemeter IGC-3, Tokyo, Japan). The difference between the radioactivity in the injected and sacrificed animal was assumed to be due to excretion, mainly urinary excretion. Blood samples were taken by cardiac puncture at sacrifice. Tissue samples of the main organs were then removed and counted in a gamma counter (Berthold). Biodistribution results were expressed as percent of injected dose per organ (% I.D./ total organ). For blood, bone, and muscle, total activity was calculated assuming that these organs constitute 6, 10, and 40% of the total weight, respectively. The remaining activity in the carcass was also measured in a dose calibrator. In vivo stability was just assessed by murine serum RP-HPLC analysis. For that, blood was centrifuged and serum separated and analyzed by HPLC after treatment with ethanol, as described above for the stability in whole human blood. RESULTS AND DISCUSSION

Preparation of [99mTc(O)(PNX)(SPh)] (X ) O, 1a; X ) S, 2a). For the introduction of stable mixed-ligand oxometal complexes with 99mTc/Re, namely those of the "3 + 1" type, with adequate physicochemical and biological characteristics for CNS receptor targeting, we have prepared oxorhenium complexes of the type [Re(O)(κ2-PNX)(κ1-SR)] (X ) O, S) anchored by the heterofunctionalized phosphines H2PNO and H2PNS, and by different monodentate thiol co-ligands (14). To check whether this type of approach could be extended to the no carrier added level, we have used complexes [99mTc(O)(PNX)(SPh)] (X ) O, 1a; X ) S, 2a) as models. These 99mTc


Fernandes et al.

Figure 1. ES/MS of [99/99mTcO(PNS/PhS)]: m/z (found) 588 (M + 1), m/z (calcd) 587 for [TcO(PNS/PhS)] (C27H23NO2PS2Tc). Scheme 2. Synthesis of the Complexes 1, 1a and 2, 2a Figure 2. Stability studies of [99mTcO(PNO/SPh)] (1a) by HPLC. In saline at 37 °C: A, after 30 min; B, after 60 min; against 1 mM glutathione solution at 37 °C: C, after 30 min; D, after 60 min;

complexes have been prepared in a one-step procedure, by direct reduction of [99mTcO4]- with stannous chloride in the presence of thiophenol and of the corresponding tridentate H2PNO or H2PNS ligands (Scheme 2). To maximize the radiochemical yield of 1a and 2a, the labeling conditions were optimized, namely the concentration, tridentate/monodentate ligand molar ratios, and pH. When dealing with H2PNS, the main radiochemical impurity was the complex [99mTc(O)(κ3-PNS)(κ1-HPNS)], formed due to the κ3,κ1-coordination versatility of PNS. This radiochemical impurity has been identified by comparing its retention time in the HPLC with that of the fully characterized analogue Re complex [Re(O)(κ3-PNS)(κ1-HPNS)] (14). Although being a less critical factor, the concentration of thiophenol also has to be controlled in order to minimize the formation of the homoleptic complex [99mTc(O)(SPh)4]-. Regarding pH, 1a requires alkaline conditions for its formation, while 2a is formed at a lower pH value (pH ∼ 6). After optimization of the labeling conditions, complexes 1a and 2a were obtained in yields higher than 90%, with only 1-3% of reduced hydrolyzed technetium and with no free pertechnetate, as indicated by TLC and HPLC. Characterization of [99mTc(O)(PNX)(SPh)] (X ) O, 1a; X ) S, 2a). The characterization of 1a and 2a was done by comparing their retention times in the HPLC (γ detection: 1a, 6.3 min; 2a, 9.1 min) with the corresponding retention times of the analogous Re complexes 1 and 2 (UV detection - 254 nm, 1: 6.3 min, 2: 9.0 min). For unambiguous identification of 2a, the complex [99/99mTc(O)(PNS)(SPh)] was synthesized using a [99mTcO4]eluate enriched with 10-8 or 10-7 mol of [99TcO4]-. As can be seen in the ES/MS spectrum shown (Figure 1) a significant peak appears at m/z 588 (M + 1), which correlates well with the value m/z of 587 calculated for [99TcO(PNS)(SPh)]. Stability and in Vivo Behavior of [99mTc(O)(PNX)(SPh)] (X ) O, 1a; X ) S, 2a). The in vitro stability of the model complexes 1a and 2a was evaluated in physiological solutions (saline, 0.01 M PBS pH 7.4, at 37 °C) and in the presence of glutathione (1 and 10 mM solutions, 37 °C). Complex 1a decomposes to more hydrophilic species during incubation in saline or PBS at 37 °C, and only

Figure 3. Stability studies of [99mTcO(PNS/SPh)] (2a) by HPLC. In saline at 37 °C: A, 30 min; B, 120 min; against 1 mM glutathione solution, at 37 °C: C, 5 min; D, 120 min.

43% or 33% of 1a remains intact after 30 or 60 min incubation, respectively (Figure 2). Challenge experiments against glutathione revealed that 1a decomposes also to more hydrophilic unknown radiochemical species even faster than in saline or PBS solutions. On the contrary, complex [99mTc(O)(PNS)(SPh)] (2a) is stable in saline, in 0.01 M PBS pH 7.4 (1:1 dilutions), and also in the presence of glutathione (1 and 10 mM solutions, 2 h, 37 °C) (Figure 3). On the basis of these results, which confirm clearly the importance of the donor atom sets on the stabilization of the "3 + 1" mixed-ligand complexes, complex 2a was chosen to pursue our studies. The overall general biodistribution profile for 2a is shown in Table 1. This complex, despite presenting a high liver uptake, also shows some initial brain uptake (0.14 ( 0.03% ID/organ, 5 min. pi), which does not decrease significantly with time (2 h after injection: 0.11 ( 0.02% ID/organ). These results, encouraged the use of the tridentate H2PNS ligand for the preparation of 99mTc mixed-ligand complexes bearing antagonists of the 5-HT1A receptors.

99mTc



Table 1. Biodistribution of 99mTcO(PNS/SPh) in Wistar Rats (n ) 5) at 5 and 120 min pi (%ID/organ ( SD) % injected dose/organ ( SD organ

5 min pi

120 min pi

brain spleen kidney heart lung liver

0.14 ( 0.03 0.95 ( 0.25 4.16 ( 0.22 0.93 ( 0.08 1.88 ( 0.16 51.30 ( 1.40

0.11 ( 0.02 0.35 ( 0.11 7.52 ( 0.41 0.46 ( 0.03 0.80 ( 0.11 26.60 ( 1.60

To achieve this goal, several thiolated arylpiperazine derivatives containing the 1-(2-methoxyphenyl)piperazine unit, a fragment of the potent and selective 5-HT1A antagonist WAY 100635 moiety, were prepared and used as monodentate co-ligands (Scheme 3). Syntheses and Characterization of the Ligands HSL2-HSL4. The ligand HSL1 was prepared as previously described (14). The monothiolates HSL2 and HSL3 were synthesized by a coupling reaction between 3-[(triphenylmethyl)thio]propionate and 1-(2-methoxyphenyl)piperazine, followed by deprotection of the thiol group or by reduction of the amide group with borane-methyl sulfide followed by deprotection, respectively. The synthesis of HSL4 involved the activation of the protected mercaptobenzoic acid with N-hydroxysuccinimide in the presence of N,N′-dicyclohexylcarbodiimide and conjugation of the resulting compound with 4-(3aminopropyl)-1-(methoxyphenyl)piperazine. Deprotection of the thiol group with triethylsilane in trifluoroacetic acid afforded HSL4. Compounds HSL2-HSL4 have been thoroughly characterized by the usual analytical techniques. The main feature of the IR spectra of HSL2 and HSL4 is the presence of a strong band at 1635 cm-1 (HSL2) and 1630 cm-1 (HSL4) assigned to the carbonyl amide stretching vibration. This band is absent in the IR spectrum of HSL3, confirming the reduction of the carbonyl group. The 1H NMR spectra of HSL2-HSL4 present all the expected resonances, including a triplet which appears at δ 2.58 in the NMR spectrum of HSL3, confirming the presence of the methylene group formed due to the reduction of the carbonyl function. In the 1H NMR spectrum of HSL2 and HSL3 also appears a triplet at 1.75 and 1.40, respectively, assigned to the thiol group. Syntheses and Characterization of the Complexes [Re(O)(PNS)(SL)] (4-6). The "3 + 1" mixedligand oxorhenium complexes (4-6) were synthesized by reacting [Re(O)Cl3(PPh3)2] with the tridentate ligand H2PNS in the presence of HSL2-HSL4, as previously described for compound 3 (Scheme 4) (14). The synthesis was carried out under nitrogen atmosphere, to avoid the oxidation of the thiol or of the phosphine ligands. The molar ratio [Re(O)Cl3(PPh3)2]/H2PNS/HSL has to be maintained at 1:1:1 to minimize the formation of the subproduct [Re(O)(κ3-PNS)(κ1-HPNS)] (14). All the complexes were fully characterized by elemental analysis, IR, 1 H and 31P NMR spectroscopy, and HPLC. The IR spectra of compounds 4-6 exhibit strong bands in the range 970-975 cm-1 assigned to the ν(RedO) stretching vibration. These values compare well with the values found for the same stretching vibration in compound 3 and also in other compounds of the "3 + 1" type stabilized by the PNS tridentate ligand and different monodentate co-ligands (970-980 cm-1) (14, 16). Another common feature to all the IR spectra is the presence of two strong absorption bands, typical of the phenylphosphine moiety, which appear in the range 745-750 and 690-700 cm-1 (22, 14). In the IR spectra of 4 and 6 also

appears a strong band at ν 1630 cm-1 and ν 1640 cm-1, respectively, assigned to the carbonyl group of the monodentate co-ligand. The 31P NMR spectra of complexes 4-6 present only one singlet, which appears in the range δ 19.6-20.0 ppm, being comparable with the values previously reported for other "3 + 1" oxo complexes with the [PNS/S] donor atom set (14-16). These resonances, assigned to the tridentate ligand H2PNS, are low-field shifted relative to the value found for the corresponding free ligand (δ -9.4 ppm), clearly indicating that the phosphorus atom is involved in the coordination to the metal. The 1H NMR spectra of complexes 4-6 reveal four sets of multiplets, integrating for one proton each, due to the methylenic protons of the H2PNS ligand, which are diastereotopic due to the asymmetry introduced in the molecule by the oxo group. The resonances due to the aromatic rings of the H2PNS and of the co-ligand appear in the expected range. Preparation of the Complexes [99mTc(O)(PNS)(SL)] (3a-6a). Complexes 3a-6a were synthesized using the procedure described for the model complex 2a. After optimizing the concentration of the tridentate ligand, 3a-6a were obtained in relatively high yields (80-95%) and were identified by comparing their retention times (γ detection) with the retention times of the well-characterized analogous rhenium complexes (3-6) (Table 2). Stability of the 99mTc Complexes (3a-6a). Complexes 3a -6a are stable in saline and in 0.01 M PBS (pH 7.4) solutions at least for 6 h (both at room temperature and at 37 °C). As mentioned above, the main drawback associated with the "3 + 1" complexes of the type [TcO(SES)(SR)] (E ) S, O, NR) is their instability toward the presence of thiolated nucleophiles, like glutathione (11-13). For the first time, we found that complexes of the "3 + 1" type (3a-6a) do not exchange with glutathione, even in the presence of concentrated solutions (1 mM and 10 mM GSH), also remaining intact in fresh rat plasma and in whole human blood (37 °C, 4 h) (Figure 4). Comparing our results with others previously reported, the high stability found for 2a-6a can only be assigned to the electronic and/or steric factors imposed by the tridentate PNS ligand. However, as referred to above, complex 1a is not stable in the presence of GSH, despite being anchored by the analogous tridentate PNO ligand. This finding led us to conclude that the stability found for complexes 2a-6a is certainly due to the electronic properties of the PNS ligand. The combination of a soft π-donor, like sulfur, and a soft π-acceptor, like phosphorus, most probably promotes a strong bond between the metal center and the sulfur atom of the coligand, leading to a less electrophilic and more stable complex. Nevertheless, a better understanding of these results would need MO theoretical calculations. Lipophilicity. The lipophilicity of all the complexes was evaluated by determination of the partition coefficient (P) in physiological conditions (n-octanol/0.1 M phosphate buffer pH 7.4). The results obtained, expressed as log Po/w, are presented in Table 2. All the compounds are lipophilic and the values found (log Po/w ) 1.70-2.01) are in the range normally accepted for the complexes to be able to cross the BBB (log Po/w ) 0.5-2.5) (3). The lipophilicity found for 3a (1.70) and for 6a (2.01) relates well with the presence of an aliphatic and an aromatic group in 3a and 6a, respectively. In Vitro Receptor Binding Studies. To evaluate the effect of the chemical modifications introduced in the monodentate ligand, and the effect of the chelate unit,


Fernandes et al.

Scheme 3. Synthesis of the Arylpiperazine Derivatives (R ) N-succinimido)

Scheme 4. Synthesis of the Re Complexes 3-6

Table 2. Properties of Compounds 3a-6a labeling compound yield (%) 3/3a 4/4a 5/5a

70-90 60-90 60-70

6/6a

85-90

tR (min) HPLC 8.2/8.1 8.3/8.1 6.3/6.1 11.8/11.7a 5.5/5.5 11.9/11.7a

log Po/w

protein binding (%) 5 min 1h

1.70 1.98 1.99

2.4 2.3 2.0

8.7 9.8 6.3

2.01

12.8

20.2

a Other HPLC conditions: column Hypersil 120 ODS (250 × 4 mm, 10 µm,1 mL/min); mobile phase methanol/0.02% Et3N in water; gradient: 50-100% methanol over 10 min followed by 10 min at 100% methanol.

the binding affinity of the complexes 3-6 for the 5-HT1A and 5-HT2A receptors was determined. Their affinity for the 5-HT1A receptor subtype was assessed in vitro on the basis of their ability to displace [3H]-8-OH-DPAT, a potent and specific 5-HT1A receptor agonist, from 5-HT1A binding sites in rat hippocampal homogenates. The IC50 values for the tested compounds are presented in Table 3. The Re complexes 3 and 6 had moderate affinity for the 5-HT1A receptors (3, IC50 120 ( 1.1 nM 6, 79.3 ( 11 nM). Substitution of the ethylenic chain of complex 3 (in HSL1) by the aromatic ring in complex 6 (in HSL4) increased the affinity for the 5-HT1A receptors. Complex 5 presents the highest affinity and selectivity for the 5-HT1A receptors (IC50 for 5-HT1A 2.35 ( 0.02 nM; competitor 5-HT2A 372 ( 11 nM). This value is similar

to the reported value for the parent compound WAY 100635 (IC50 ) 2.2 nM), suggesting that the affinity of the o-methoxyphenylpiperazine moiety for 5-HT1A receptors is not reduced by its functionalization with a thiol group and by the presence of the [Re(O)(PNS)]+ metal fragment (18). A further important criterion for the potential application of this type of biocomplexes is the selectivity of the pharmacophore for the 5-HT1A receptors. According to Halldin et al., ideally the affinity of a radioligand should be highest for the site of interest by more than 1 order of magnitude (23). Compound 5 presents a selectivity toward the 5-HT2A receptors which is over 150-fold. Complex 4 completely lost affinity for the 5-HT1A receptors, must probably due to the presence of the carbonyl function in the monodentate ligand, which seems to prevent the access to the receptor-binding site. Biodistribution. The in vivo behavior of the 99mTc complexes (3a-6a) was evaluated in mice at 5 and 60 min post intravenous injection. Figure 5 shows the tissue distribution results expressed as % I.D./total organ in the most relevant organs. These results indicate a similar pattern for all the complexes under study, showing a slow clearance from organs such as blood and muscle and a slow rate of total radioactivity excretion from whole animal body (less than 12% of total injected dose 1 h after administration). All the complexes have high initial blood and muscle uptake that slowly clear, mainly via the hepatobiliary pathway. Complex 4a presents a quite fast blood and muscle clearance when compared to 3a, 5a, and 6a. As expected for lipophilic compounds, there is a very high and rapid liver uptake that decreases over the time with a significant amount of injected dose cleared into the intestines. The low kidney uptake associated to the low total radioactivity excretion indicates that those complexes are mainly eliminated through the hepatobiliary tract with quite low urinary elimination. Despite being neutral and lipophilic, all the complexes (3a-6a) present low brain uptake (%I.D./organ e 0.2%, 5 min after administration) and no significant retention of the radioactivity is observed. HPLC analysis of the murine serum collected after 5 min and 1 h postinjection indicated that all the complexes remain intact in circulation, confirming their high in vivo stability. Despite the good perspectives related to the charge, lipophilicity, and in vitro and in vivo stability of the complexes, they are not able to cross the BBB, which would enable the in vivo assessment of 5-HT1A receptors.

99mTc



Figure 4. HPLC chromatograms of 6a (A); (B) after incubation with rat serum, 37 °C, 4 h; (C) after incubation with 10 mM gluthathione solution, 2 h, 37 °C. Table 3. IC50 (nM) Values for the Tested Compounds 3a-6a IC50 (nM) complex

5-HT1A ([3H]OH-DPAT)

5-HT2A ([3H]-ketanserin)

5-HT2A/5HT1A

3a 4a 5a 6a

120 (1.1 15170 ( 532 2.35 ( 0.02 79.3 (1.2

860 ( 51 148 ( 50 372 ( 11 542 ( 15

7.16 < 0.01 158.3 6.8

exchange with glutathione was observed, even when high concentrations of this nucleophile are used. This is a remarkable feature for this type of complex, and these are the first examples of "3 + 1" complexes presenting no transchelation toward glutathione. One of the complexes (5/5a) also showed a remarkable affinity and selectivity for the 5-HT1A receptors (IC50 for 5-HT1A 2.35 ( 0.02 nM; competitor 5-HT2A 372 ( 11 nM; 5-HT1A/5HT2A ) 158.3), comparable to the corresponding values found for the [11C]-WAY 100635 (IC50 for 5-HT1A 2.2 nM). These results confirmed the possibility of preparing metal complexes without affecting the affinity and selectivity of the biomolecules for the corresponding receptors. Unfortunately, despite displaying adequate characteristics, such as neutrality, high lipophilicity, and high in vitro and in vivo stability, none of the complexes cross the BBB, as demonstrated by biodistribution studies in mice. This topic is an open field and deserves considerable attention. Such work is of the highest importance, when aiming at the development of brain-related metallo(radio)pharmaceuticals. ACKNOWLEDGMENT

This work has been partially supported by a bilateral project ICCTI/DAAD and by COST Action B12. LITERATURE CITED Figure 5. Biodistribution results in mice (% injected dose/ organ) for complexes 3a-6a.

This can be due to the presence of the chelate moiety that influences the properties of the whole molecule in an unclear manner. Finding rules for or, better, relations between structure parameters of metal complexes to allow passage through the BBB is an important goal but has hardly been investigated so far. Attempts to interpret the BBB transport of Tc complexes on the basis of rough physicochemical parameters such as lipophilicity and basicity have not been successful. CONCLUDING REMARKS

We were able to prepare the "3 + 1" oxotechnetium(V) complexes 1a and 2a at the nca level, using the heterofunctionalized phosphines H2PNO and H2PNS as tridentate ligands, and thiophenol as monodentate co-ligand. These model complexes present different in vitro stability, confirming the importance of the donor atom set. Using the tridentate H2PNS ligand, several "3 + 1" oxotechnetium(V) complexes bearing 5-HT1A receptorbinding ligands have been prepared in good yields and high radiochemical purity (>95%). All are stable in saline, PBS pH 7.4, rat plasma, and human serum. No

(1) Hom, R. K., and Katzenellenbogen, J. A. (1997) Technetium99m-Labeled Receptor-Specific Small-Molecule Radiopharmaceuticals: Recent Developments and Encouraging Results. Nucl. Med. Biol. 24, 485-498. (2) Jurisson, S. S., and Lydon, J. D. (1999) Potential Technetium Small Molecule Radiopharmaceuticals. Chem. Rev. 99, 22052218. (3) Johannsen, B., and Pietzsch, H. (2002) Development of technetium-99m-based CNS receptor ligands: have there been any advances?. Eur. J. Nucl. Med. 29, 263-275. (4) Meegalla, S., Plossl, K., Kung, M. P., and Stevenson, D. A. (1995) First example of a 99mTc Complex as a Dopamine Transporter Imaging Agent. J. Am. Chem. Soc. 117, 1103711038. (5) Kung, H. F., Kim, H.-J., Kung, M. P., Meegalla, S., Plossl, K., and Lee, H.-K. (1996) Imaging of dopamine transporters in humans with technetium-99m TRODAT-1. Eur. J. Nucl. Med. 23, 1527-1530. (6) Spies, H., Fietz, T., Pietzch, H.-J., Johannsen, B., Leibnitz, P., Reck, G., Scheller, D., and Klostermann, K. (1995) Neutral Oxorhenium(V) Complexes with Tridentate Dithiolates and Monodentate Alkane- or Arene-thiolate Coligands. J. Chem. Soc., Dalton Trans. 2277-2280. (7) Johannsen, B., Scheunemann, M., Spies, H., Brust, P., Wober, J., Syhre, R., and Pietzch, H.-J. (1996) Technetium(V) and Rhenium(V) Complexes for 5-HT2A Serotonin Receptor Binding: Structure-Affinity Considerations. Nucl. Med. Biol. 23, 429-438.

668 Bioconjugate Chem., Vol. 16, No. 3, 2005 (8) Mastrostamis, S. G., Papadopoulos, M. S., Pirmettis, I. C., Paschali, E., Varvarigou, A. D., Stassinopoulou, C. I., Raptopoulou, C. P., Terzis, A., and Chiotellis, E. (1994) Tridentate Ligands Containing the SNS Donor Atom Set as a Novel Backbone for the Development of Technetium Brain-Imaging Agents. J. Med. Chem. 37, 3212-3218. (9) Papagiannopoulou, D., Pirmettis, I., Maina, T., Pelecanou, M., Nikolopoulou, A., Chiotelis, E., Raptopoulou, C. P., Vlahos, A. T., Terzis, A., Papadopoulos, M., and Chiotelis, E. (2001) Development of novel mixed-ligand oxotechnetium [SNS/S] complexes as potencial 5-HT1A receptor imaging agents. J. Biol. Inorg. Chem. 6, 256-265. (10) Fernandes, C., Patrı´cio, L., Moreira, R., Cantinho, G., Pena, H., Campello, P. C., and Santos, I. (2004) Novel 3+1 MixedLigand Technetium-99m Complexes Carrying Dipeptides as Monodentate Ligands. Nucl. Med. Biol. 31, 139-145. (11) Gupta, A., Seifert, S., Syhre, R., Scheunemann, P., Brust, P., and Johannsen, B. (2001) Reactivity of 99mTc(V) "3 + 1" mixed-ligand complexes towards glutathione. Radiochim. Acta 89, 43-49. (12) Syhre, R., Seifert, S., Spies, H., Gupta, A., and Johannsen, B. (1998) Stability versus reactivity of "3 + 1" mixed-ligand technetium-99m complexes in vitro and in vivo. Eur. J. Nucl. Med. 25, 793-796. (13) Seifert, S., Gupta, A., Syhre, R., Spies, H., and Johannsen, B. (2001) Ligand-exchange reaction of labile “3 +1” 99mTc(V) complexes with SH group-containing proteins. Appl. Radiat. Isot. 54, 637-644. (14) Correia, J. D. G., Domingos, A., Santos, I., and Spies, H. (2001) Synthesis and characterization of mixed-ligand oxorhenium(V) complexes with new [(PNO/S)/(S)] donor atom sets. J. Chem. Soc., Dalton Trans. 2245-2250. (15) Correia, J. D. G., Domingos, A., and Santos, I. (2000) Neutral Trichlorooxorhenium(V) complexes containing phosphane ligands of the type PN2 and PNO. Eur. J. Inorg. Chem. 1523-1529. (16) Kniess, T., Fernandes, C., Santos, I., Spies, H., and Kraus, W. (2003) Silylated mixed-ligand rhenium complexes with the [PNS/S] donor atom set. Inorg. Chim. Acta 348, 237-241.

Fernandes et al. (17) Passchier, J., and Waarde, A. V. (2001) Visualisation of serotonin-1A (5-HT1A) receptors in the central nervous system. Eur. J. Nucl. Med. 28, 113-129. (18) Pike, V. W., Halldin, C., Wikstrom, H., Marchais, S., McCarron, J. A., Sandell, J., Nowicki, B., Swahn, C.-G., Osman, S., Hume, S. P., Constantinou, M., Anfre´e, B., and Farde, L. (2000) Radioligands for the study of brain 5-HT1A receptors in vivo-development of some new analogues of WAY. Nucl. Med. Biol. 27, 449-455. (19) Brenner, D., Davison, A., Lister-James, J., and Jones, A. G. (1984) Synthesis and characterization of a series of isomeric oxotechnetium(V) diamido dithiolates. Inorg. Chem. 23, 3793-3797. (20) Alberto, R., Schibli, A., Egli, A., Schubiger, P., Herrmann, W. A., Artus, G., Abram, U., and Kaden, T. A. (1995) Metal carbonyl synthesis XXII. Low-pressure carbonylation of [MOCl4]- and [MO4]-: the Technetium(I) and Rhenium(I) complexes [NEt4][MCl3(CO)3]. J. Organomet. Chem. 493, 119-127. (21) Ram, M. S., and Hupp, J. T. (1991) Generalized synthesis of cis- and trans-dioxorhenium(V) (bi)pyridyl complexe. Inorg. Chem. 30, 130-133. (22) Nakamoto, K. (1970) Infrared Spectra of Inorganic and Coordination Compounds, 2nd ed., pp 205-209, Chapter 3. Wiley-Interscience, New York. (23) Halldin, C., Gulya´s, B., Langer, O., and Farde, L. (2001) Brain radioligands - State of the art and new trends. Q. J. Nucl. Med. 45, 139-152. (24) Fernandes, C., Correia, J. D. G., Gano, L., Santos, I., Seifert, S., Spies, H., and Syhre, R. (2001) Tc oxocomplexes with the PNO/S and PNS/S donor atom sets: labelling of a 5HT1A receptor-binding ligand. J. Labelled Comp. Radiopharm. 44, S518-S520.

BC049718K

Dramatic Effect of the Tridentate Ligand on the Stability of 99mTc "3 +

Recommend Documents