Chemical and Biological Characterization of Technetium(I

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Bioconjugate Chem. 2000, 11, 414−424

Chemical and Biological Characterization of Technetium(I) and Rhenium(I) Tricarbonyl Complexes with Dithioether Ligands Serving as Linkers for Coupling the Tc(CO)3 and Re(CO)3 Moieties to Biologically Active Molecules H.-J. Pietzsch,*,† A. Gupta,† M. Reisgys,† A. Drews,† S. Seifert,† R. Syhre,† H. Spies,† R. Alberto,‡ U. Abram,§ P. A. Schubiger,‡ and B. Johannsen† Forschungszentrum Rossendorf, Institut fu¨r Bioanorganische und Radiopharmazeutische Chemie, PF 510119, D-01314 Dresden, Germany, Paul-Scherrer-Institut Villigen, Abteilung fu¨r Radiopharmazie, CH-5232 Villigen, Switzerland, and Forschungszentrum Rossendorf, Institut fu¨r Radiochemie, PF 510119, D-01314 Dresden, Germany. Received November 22, 1999

The organometallic precursor (NEt4)2[ReBr3(CO)3] was reacted with bidendate dithioethers (L) of the general formula H3C-S-CH2CH2-S-R (R ) -CH2CH2COOH, CH2-CtCH) and R′-S-CH2CH2S-R′ (R′ ) CH3CH2-, CH3CH2-OH, and CH2COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)3L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO3)(H3C-S-CH2CH2-S-CH2CtCH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH2CH2-SCH2CH2-S-CH2CH2-OH was reacted with the trinitrato precursor [Re(NO3)3(CO)3]2- to yield a cationic complex [Re(CO)3(HO-CH2CH2-S-CH2CH2-S-CH2CH2-OH)]NO3 Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H3C-S-CH2CH2-S-CH2CH2-COOH and HOOC-CH2-S-CH2CH2-S-CH2-COOH with (NEt4)2[ReBr3(CO)3] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)3(OOC-CH2-S-CH2CH2-S-CH2-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The nocarrier-added preparation of the analogous 99mTc(I) carbonyl thioether complexes could be performed using the precursor fac-[99mTc(H2O)3(CO)3]+, with yields up to 90%. The behavior of the chlorine containing 99mTc complex [99mTcCl(CO)3(CH3CH2-S-CH2CH2-S-CH2CH3)] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [99mTc(H2O)(CO)3(CH3CH2-S-CH2CH2-S-CH2CH3)]+ occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [99mTcCl(CO)3(CH3CH2-S-CH2CH2-S-CH2CH3)] Tc1 and [99mTcCl(CO)3(CH2-S-CH2CH2-S-CH2-CtCH)] Tc2 a significant initial brain uptake (1.03 ( 0.25% and 0.78 ( 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.

INTRODUCTION

The γ-emitting nuclide technetium-99m plays the dominating role in diagnostic nuclear medicine because of its optimal nuclide properties. Either as nonradioactive surrogate of technetium or because of the favorable properties of rhenium-186 and rhenium-188 for therapy, the congener rhenium is usually included in the consideration of the radiopharmaceutical chemistry of techne* To whom correspondence should be addressed. Fax: (0351) 260 3232. E-mail: [email protected]. † Forschungszentrum Rossendorf, Institut fu ¨ r Bioanorganische und Radiopharmazeutische Chemie. ‡ Paul-Scherrer-Institut Villigen. § Forschungszentrum Rossendorf, Institut fu ¨ r Radiochemie.

tium. A number of reviews covering this field have appeared recently (1-3). Current development of Tc-99m radiopharmaceuticals considers the present needs for new and broadly applicable tracers for metabolism and receptor studies. The strategy to design such technetiumbased compounds involves incorporation of the radionuclide into biologically active molecules, which requires chelating of the technetium at an appropriate oxidation state and linking the chelate to the biomolecule. Advances in this field depend on the availability of chelating systems that are well-suited to be combined with the biological “anchor group”. In this sense, the chelates should distinguish themselves by high stability, small size, adoptable lipophilicity, and the possibly absence of isomers (4-6).

10.1021/bc990162o CCC: $19.00 © 2000 American Chemical Society Published on Web 03/31/2000

Characterization of Technetium(I) and Rhenium(I) Complexes

In the past years, various concepts for chelating ligands have been outlined. The most successful of them are focused on oxotechnetium(V) complexes (7) containing the [TcdO]3+ moiety, which is readily accessible by reduction of pertechnetate in the presence of suitable chelators such as tetradentate N2S2 ligands (8-12) or mixtures of ligands according to the so-called “3+1” concept (13-18). However, the oxo core, usually square-pyramidal coordinated and with a free position trans to the oxo ligand, is a quite polar unit. The stricter that the requirements are for specific agents, the more important the question as to whether such a polarity is beneficial or not. Alternatives are the oxo-free, lower oxidation states, namely, +3 and preferably +1. The rarely exploited oxidation state +1 came into the limelight by the discovery of myocardium affine hexakis isonitriles of technetium(I) and the successful clinical application of 99mTc methoxy-isobutyl isonitrile (19). Despite this progress, the oxidation state +1 has been addressed only in a minor way (20-24) before a new most promising approach to low valent, inert technetium compounds on basis of Tc(I) carbonyl complexes has been elaborated (25-27). Intensive investigations in the Tc carbonyl chemistry made available an organometallic Tc(I) aqua ion, [Tc(H2O)3(CO)3]+, from TcO4- under normal pressure, the prerequisite to exploit the small [Tc(CO)3]+ moiety for the labeling of biomolecules is given (28, 29). It could be shown that isonitriles (30), thiourea and its derivatives (31), thiophosphoryl amides (32), cyclopentadiene (33), various N-containing ligands, such as histamine, imidazole, histidine, and Schiff bases (34, 35), and water-soluble phosphines (36) are able to easily react with the [Tc(H2O)3(CO)3]+ to yield stable complexes. Double ligand-transfer reactions developed by Spradau et al. give access to derivatized cyclopentadienyltricarbonyl technetium and rhenium complexes for labeling steroids (37, 38). Thioether ligands may serve in an alternative approach to fully exploit the potential of the [M(CO)3]+ moiety (M ) Tc, Re) for the design of radiotracers. Thioethers with the π-acceptor properties of the sulfur show a great potency to coordinate at the metal(I) carbonyl center and a couple of Re carbonyl complexes with thioether ligands are known (29, 39-42). Recently, we have published the synthesis of Tc and Re dithioether carbonyl complexes bearing steroids as biologically active anchor group (43, 44) as well as tropanol-functionalized Tc thioether carbonyls to label the dopamine transporter (45). In the present paper, we describe the basic chemistry of the Tc(I)/Re(I) carbonyl thioethers as relevant for the radiotracer design. This involves the synthesis and structural analysis of rhenium complexes with functionalized bidentate thioether ligands derived from (NEt4)2[ReBr3(CO)3] and (NEt4)2[Re(NO3)3(CO)3]. The preparation of the analogous 99mTc complexes is described as well as their stability in aqueous solution and in plasma, and their biodistribution in rats. EXPERIMENTAL PROCEDURES

General. All chemicals and solvents were of reagent grade and used without further purification. The thioether ligands L1-L5 were synthesized according to standard procedures and to Beger et al. (46). (NEt4)2[ReBr3(CO)3] was prepared as published elsewhere (47). (NEt4)2[Re(NO3)3(CO)3] was obtained by reaction of (NEt4)2[ReBr3(CO)3] with a 3-fold equiv of silver nitrate in water (27).

Bioconjugate Chem., Vol. 11, No. 3, 2000 415

Elemental analyses were performed on a LECO Elemental Analyzer CHNS-932. Melting points were obtained on a BOE ¨ TIUS-Mikroheiztisch and are uncorrected. IR spectra were measured as KBr pellets on a PerkinElmer FTIR-spectrometer SPECTRUM 2000. 1 H NMR spectra (400 MHz) were recorded on a Varian spectrometer INOVA-400. The Re complexes and the analogous 99mTc compounds were identified and characterized by gradient HPLC (Hypersil column, Knauer, 10 µm, 250 mm × 4 mm), methanol/phosphate buffer (0.01 M, pH 7.4) from 50% methanol to 100% within 5 min, 1 mL/min. Cationic 99mTc and Re species were additionally characterized by paper electrophoresis (400 V, 20 V/cm, 50 min; FN 4 paper (Niederschlag), acetonitrile/0.1 M phosphate buffer (50/50 v/v), pH 7.4. Further 99mTc species were characterized by capillary electrophoresis using a 3DCE device (Hewlett-Packard) equipped with a diode array detection (DAD) system and radioactivity detection as published by Jankowsky et al. (48). A fused silica capillary with inner diameter of 50 µm and an effective length of 64 cm was employed. For running electrolyte, phosphate buffer 50 mM mixed with 50% of acetonitrile was used. During runs, voltages of 25 kV were applied, and for investigations at a pH of 9.3, an additional external air pressure of 50 mbar was applied. X-ray Data Collection and Processing. The intensities for the X-ray structure determination were collected on an automated single-crystal diffractometer of the type CAD4 (Enraf-Nonius) MoKR (Re2) or CuKR (Re6, Re8) radiation, respectively, with ω scans. The cell dimensions were determined by the angular setting of 25 high-angle reflections. The structures were solved by heavy-atom Patterson synthesis using SHELXS-86 (49). Refinement was performed with SHELXL-93 (50). All non-hydrogen atoms were located from successive Fourier maps and refined with anisotropic thermal parameters. In the case of complex Re8, the hydrogen atoms were fully refined, whereas for Re2 and Re6 the hydrogen atoms were placed in calculated positions and refined using the “riding model” option of SHELXL-93. A summary of the crystallographic data is given in Table 1. More details of the structure determination have been deposited with the Cambridge Crystallographic Data Centre under the deposit numbers CCDC 134865 (RE2), CCDC 134866 (Re6), and CCDC 134867 (Re8). General Synthesis Procedure for the Complexes [ReBr(CO3)L] (Re1) - (Re5, Re7). A total of 130 µmol of the appropriate ligand L1-L5, dissolved in 1 mL of methanol was added to a stirred solution of 100 mg (130 µmol) (NEt4)2[ReBr3(CO)3], dissolved in 1 mL of methanol. After stirring the reaction mixture at ambient temperature for 1 h, the solvent was removed to dryness in a vacuum. The remaining residue was taken up in dry THF and the white precipitate (NEt4Br) was filtered off. The filtrate was dried and washed three times with water and diethyl ether until a white solid was formed. After each washing procedure, the residue was dried in a vacuum. Bromo(3,6-dithiaoctane-S,S)tricarbonylrhenium(I)] (Re1). Yield: 57 mg (88%). HPLC: Rt ) 7.6 min. mp ) 90-92 °C. IR (KBr) (cm-1): 2024 (CO), 1932 (CO), 1900 (CO). Anal. (C9H14BrO3ReS2) calcd: C, 21.60; H, 2.82; S, 12.81; Br, 15.97%. Found: C, 21.68; H, 2.66; S, 12.97; Br, 15.43%. [Bromo(4,7-dithia-1-octyne-S,S)tricarbonylrhenium(I)] (Re2). Colorless crystals of Re2 suitable for X-ray diffraction were obtained from acetone/n-hexane.

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Table 1. Crystallographic Data for X-ray Diffraction Studies of Re Complexes Re2, Re6, and Re8 formula fw crystal system space group a (Å) b (Å) c (Å) R (deg) β (deg) γ (deg) V (Å3) temp (K) d (g/cm3) µ (mm-1) absorbance correction Tmin/Tmax F(000) λ (Å), radiation crystal size (mm3) 2θ-range hkl no. of coll. reflns no. of indep. reflns/Rint no. of obsd reflns no. of parameters GOF R [I > 2s(I)] R1 wR2 R (all data) R1 wR2

Re2

Re6

Re8

C9H10BrO3ReS2 496.40 monoclinic C2/c 26.995 (5) 7.892 (1) 13.460 (3) 90 108.69 (1) 90 2716.4 (9) 8 293(2) 2.428 12.184 ψ scans 0.7532/0.9716 1840 0.710 73 MoKR 0.30 × 0.15 × 0.15 3.01-24.99 0 e h e 34 0 e k e 10 -17 e l e 16 2453 2401/0.0393 1793 145 1.073

C9H9O7ReS2 479.48 monoclinic P2(1)/cP 8.615 (1) 10.630 (1) 13.899 (1) 90 100.17(1) 90 1252.8(2) 4 213(2) 2.542 22.376 ψ scans 0.3808/0.9632 904 1.541 84, CuKR 0.2 × 0.2 × 0.1 5.22 to 64.95 -1 e h e 10 0 e k e 12 -16 e l e 16 2594 2129/0.0296 2082 208 1.104

C9H14NO8ReS2 514.53 triclinic P1 h 8.870 (1) 11.323 (2) 16.565 (3) 105.78 (1) 98.47 (1) 102.66 (1) 1523.7 (4) 4 293(2) 2.243 18.524 ψ scans 8.8367/0.9985 984 1.541 84, CuKR 0.30 × 0.05 × 0.05 5.24-64.95 -1 e h e 10 -13 e k e 13 -19 e l e 19 6071 4969/0.0445 4368 380 1.079

0.0467 0.1014

0.0355 0.0984

0.0381 0.1023

0.0760 0.1185

0.0365 0.1030

0.0446 0.1076

Yield: 49 mg (80%). HPLC: Rt ) 7.2 min. mp ) 95-96 °C. IR (KBr) (cm-1): 2032 (CO), 1940 (CO), 1900 (CO). Anal. (C9H10BrO3ReS2) calcd: C, 21.78; H, 2.03; S, 12.92; Br, 16.10%. Found: C, 21.85; H, 2.09; S, 12.94; Br, 15.10%. [Bromo(1-carboxy-3,6-dithiaheptane-S,S)tricarbonylrhenium(I)] (Re3). Yield: 56 mg (81%). HPLC: Rt ) 3.2 min. mp ) 157-158 °C. IR (KBr) (cm-1): 2032 (CO), 1916 (CO), 1712 (CdO). Anal. (C9H12BrO5ReS2) calcd: C, 20.38; H, 2.27; S, 12.09; Br, 15.06%. Found: C, 20.60; H, 2.56; S, 11.97; Br, 14.17%. [Bromo(1,6-dicarboxy-2,5-dithiahexane-S,S)tricarbonylrhenium(I)] (Re4). Yield: 57 mg (88%). mp ) 90-92 °C. IR (KBr) (cm-1): 2024 (CO), 1932 (CO), 1900 (CO). Anal. (C9H10BrO7ReS2) calcd: C, 19.29; H, 1.78; S, 11.43; Br, 14.28%. Found: C, 19.48; H, 1.87; S, 11.28; Br, 14.51%. [1-Carboxylato-3,6-dithiaheptane-O,S,S)tricarbonylrhenium(I) (Re5). A total of 59 mg (130 µmol) of 1-carboxy-3,6-dithiaheptan (L3) was added to 130 µL NaOH (1 N) in 1 mL of water. The reaction mixture was stirred until the ligand was dissolved completely. To this solution was added 100 mg (130 µmol) of (NEt4)2[ReBr3(CO)3] in 1 mL of water. After a reaction time of 1 h, the water was removed by vacuum and the residue was washed with a small amount of water and filtrated. The white powder was washed with diethyl ether to yield 31 mg (53%). HPLC: Rt ) 6.5 min. mp ) 85 °C. IR (KBr) (cm-1): 2032 (CO), 1940 (CO), 1900 (CO), 1632 (CdO). Anal. (C9H11O5ReS2) calcd: C, 24.05; S, 2.47; H, 14.26%. Found: C, 24.13; H, 3.00; S, 14.69%. [(1-Carboxylato-6-carboxy-2,5-dithiahexane-O,S,S)tricarbonylrhenium(I)] (Re6). A total of 81 mg (390 µmol) of 2,5-dithiahexane-1,6-dicarboxylic acid was dissolved in 5 mL of water. A total of 300 mg (390 µmol) (NEt4)2[ReBr3(CO)3] and 1 equiv of 1 N NaOH was added

to the clear solution, which became turbid after 15 min. A white precipitate formed over a further hour of stirring. The precipitate was filtered to yield 150 mg (83%) of analytically pure compound Re6. IR (KBr) (cm-1): 2032 (CO), 1934 (CO), 1892 (CO). Anal. (C9H9O7ReS2) calcd: C: 22.54, H: 1.89%. Found: C, 22.33; H, 1.92%. [Bromo(1,8-dihydroxy-3,6-dithiaoctane-S,S)tricarbonylrhenium(I)] Re7. Yield: 57 mg (83%). mp ) 90-92 °C. IR (KBr) (cm-1): 2024 (CO), 1932 (CO), 1900 (CO). Anal. (C9H14BrO5ReS2) calcd: C, 20.30; H, 2.63; S, 12.03; Br, 15.04%. Found: C, 20.18; H, 2.47; S, 12.28; Br, 15.33%. [(1,8-Dihydroxy-3,6-dithiaoctane-O,S,S)tricarbonylrhenium(I)]nitrate (Re8). A total of 24 mg (130 µmol) of 1,8-dihydroxy-3,6-dithiaoctane (L5), dissolved in 1 mL of methanol, was added to a stirred solution of 93 mg (130 µmol) (NEt4)2[Re(NO3)3(CO)3] in 1 mL of water. After 10 min, the solvent was removed and the residue dissolved in dry THF. The precipitate was filtered off. Removing the solvent from the filtrate yields 59 mg (88%) of a white precipitate. Colorless needles for X-ray analysis were obtained from chloroform/n-hexane. mp ) 119122 °C. IR (KBr) (cm-1): 2032 (CO), 1936 (CO), 1384 (NO3). Anal. (C9H14NO8ReS2) calcd: C, 21.01; H, 2.74 N 2.72; S, 12.46%. Found: C, 21.36; H, 3.01 N 2.90; S, 12.40%. General Synthesis Procedure for the Preparation of [99mTc(H2O)3(CO)3]+ According to Ref 34. Na2CO3 (0.038 mmol) and NaBH4 (0.26 mmol) were added to a 10 mL vial, which was closed and flushed for 10 min with CO. A 1.5 mL sample of a 99mTc generator eluate mixed with 0.25 mL of propylene glycol (containing up to 200 MBq Na[99mTcO4] in saline) was added, and the solution heated to 75 °C for 40 min. Quality control was performed by gradient HPLC or TLC (silica gel, methanol/HCl (95/5 v/v, Rf 0.4). Yield: 90-95%.


[Chloro(3,6-dithiaoctane-S,S)tricarbonyltechnetium(I)] (Tc1). A total of 400 µL of 1 N HCl for adjusting the pH to 1, 0.25 mL of propylene glycol, 0.25 mL of methanol, and 30 mg of NaCl were added to the solution of the [99mTc(H2O)3(CO)3]+ precursor. 3,6-Dithiaoctane (L1) (1.0 µmol), dissolved in methanol, was added, and the reaction mixture was heated to 70 °C for 15 min. Finally, the reaction mixture was neutralized with 0.1 N NaOH. HPLC: Rt ) 7.6 min (yield, 70%). For separation of by-products and ligand excess, the reaction solution was given onto a semipreparative Hypersil column (250 × 8 mm, 10 µm, flow rate 2.0 mL/ min) and eluted using a linear gradient system [t (min)/% A]: 5/50, 10/100, and 15/100 of methanol (A)/0.01 M phosphate buffer of pH 7.4 (B). Immediately after separation, the complexes were diluted 1:1 with 0.9% NaCl containing 25% propylene glycol, and the methanol was removed by evaporation to yield a stable solution for biodistribution studies. For stability studies, the complexes were alternatively diluted after separation in PBS, in H2O, or in a solution of 10 mM GSH in water. [Chloro(4,7-dithia-1-octyne-S,S)tricarbonyltechnetium(I)] (Tc2). A total of 400 µL of 1 N HCl for adjusting the pH to 1, 0.25 mL of propylene glycol, 0.25 mL of methanol, and a further 30 mg of NaCl were added to the solution of the [99mTc(H2O)3(CO)3]+ precursor. 4,7Dithia-1-octyne (L2) (3.3 µmol), dissolved in methanol, was added, and the reaction mixture was heated to 70 °C for 15 min. Finally the reaction mixture was neutralized with 0.1 N NaOH. HPLC: Rt ) 7.2 min (yield, 70%). The separation of Tc2 followed the procedure as described for Tc1. [Chloro(1-carboxy-3,6-dithiaheptane-S,S)tricarbonyltechnetium(I)] (Tc3). The preparation was carried out as described for Tc1, but after preparation, the solution was not neutralized. The separation of complex Tc3 was carried out with a semipreparative Hypersil column (250 × 8 mm, 10 µm, flow rate 2.0 mL/min) and eluted using a linear gradient system [t (min)/% A]: 5/20, 6/100, and 10/100 of methanol (A)/0.01 M phosphate buffer of pH 7.4 (B). To avoid the conversion of complex Tc3 to Tc5, the eluate was given into a saturated solution of NaCl at pH 2 immediately after separation. [Chloro(1,6-dicarboxy-2,5-dithiahexane-S,S)tricarbonyltechnetium(I)] (Tc4). The preparation was carried out as described for Tc3, but the gradient system used for purification was modified as follows [t (min)/% A]: 5/0, 5/100, and 10/100 of methanol (A)/0.01 M phosphate buffer of pH 7.4 (B). [1-Carboxylato-3,6-dithiaheptane-O,S,S)tricarbonyltechnetium(I) (Tc 5). The preparation was carried out as described for Tc1, but after preparation, the pH of the solution was adjusted to pH 8. After standing for about 30 min at room temperature, Tc5 was separated using the HPLC conditions as described for complex Tc3. [(1-Carboxylato-6-carboxy-2,5-dithiahexane-O,S,S)tricarbonyltechnetium(I)] (Tc6). The preparation was carried out as described for Tc1 but after preparation, the pH of the solution was adjusted to pH 8. After standing for about 30 min at room temperature, Tc6 was separated using the HPLC conditions as described for complex Tc4. Determination of Partition Coefficients. Partition coefficients of the 99mTc complexes were determined by shakeflask method by mixing 0.5 mL of the complex solution (0.9% NaCl solution and 20% propylene glycol) and 0.5 mL of octanol and shaking for 5 min. After centrifugation,


aliquots of both phases were counted and the partition coefficient calculated. Stability Studies in Solution. 99mTc complexes dissolved in MeOH were diluted with an equivalent volume of phosphate buffer 0.1 M, 0.9% NaCl, or H2O containing 25% propylene glycol immediately after HPLC separation. At various incubation time, the samples were analyzed by HPLC with a Hypersil ODS column (250 × 4 mm) using a linear gradient system [t (min)/% A]: 5/50, 5/100, and 10/100 of methanol (A)/0.01 M phosphate buffer of pH 7.4 (B). Ligand Exchange (Challenge) Experiments with Glutathione (GSH). 99mTc complexes dissolved in MeOH were diluted with an equivalent volume of an aqueous solution of 20 mM GSH containing 25% of propylene glycol immediately after HPLC separation, resulting in a final glutathione concentration of 10 mM. The analyses were performed by HPLC as described above. Stability Studies in Plasma. A total of 50 µL of the appropriate 99mTc complex solution (0.9% NaCl, 12% propylene glycol) was incubated in 200 µL of rat plasma at 37 °C. After an incubation time of 30 min, the samples were analyzed by HPLC with a Supelguard column (20 × 4.6 mm, 10 µm, flow rate 1.0 mL/min) using a linear gradient 95% A to 40% A in 15 min [A, 2-propanol/0.1% trifluoroacetic acid (TFA) (10/90); B, 2-propanol/0.1% TFA (90/10)]. Alternatively, the HPLC analyses were carried out with a PRP-3 column (Hamilton, 150 × 4 mm, 10 µm, flow rate 1.0 mL/min) using a linear gradient system [t (min)/% B]: 5/0, 10/70, and 5/70 of 10 mM phosphate buffer (PBS) of pH 7.4 (A) and acetonitrile (B). Biodistribution Studies. The animal studies in male Wistar rats (5-6 weeks old) were carried out according to the relevant national regulations. A total of 500 µL of 99m Tc complex solution (saline, propylene glycol 25%) was injected into the tail vein of rats. After the injection, the rats were sacrificed by heart puncture under ether anaesthesia 5 and 120 min p.i.. Selected organs were isolated for weighing and counting. The accumulated radioactivity in the tissue of organs were calculated in terms of percentage of injected dose per organ as well as percent injected dose per gram blood. RESULTS AND DISCUSSION

Rhenium Chemistry. The dithioether ligands L1 to L5 listed in Scheme 1 are able to bind to the Re(I) core. Except for L1, the ligands are equipped with functional groups such as hydroxy, carboxylic, and propargylic groups to bind a biomolecule. Scheme 1


Pietzsch et al.

Scheme 2

Consequently, only the two sulfur atoms and the three carbonyl ligands are involved beside the remaining bromo ligand. The attachment of the bromotricarbonyl rhenium(I) core through the dithioether sulfur leads to a rhenium(I) chelate in which the metal is a stereogenic center. Furthermore, the coordination of the prochiral thioether sulfur also results in the formation of stereogenic sulfur donor atoms. The formation of stereoisomers during the complexation reflects a general feature of the dithioethercarbonyl design when a nonsymmetrical dithioether is used. So the complexes Re2, Re3, Re5, and Re6 occur as isomeric mixtures. The infrared spectra of Re1-Re4 and Re7 show strong absorption bands at 2032, 1940, and 1900 cm-1, indicating an approximate CS symmetry of the complexes (51). 1H NMR spectra are negligible because of the overlap of methylene protons leading to complicated signal patterns.

The rhenium complexes have been synthesized as fully characterized surrogates for the analogous 99mTc complexes prepared at the no-carrier-added level. Coinjections of couples of Re and 99mTc compounds into HPLC under various elution conditions should confirm the structural similarity of both substances. The mixed halide/carbonyl complex (NEt4)2[ReBr3(CO)3] proved to be a very useful starting material and was used as the most convenient source of the “fac-Re(CO)3” fragment in the reaction with thioether ligands. In solution, a complete exchange of the three bromine ligands by solvent molecules was observed (25-27). The reaction of the resulting species [Re(CO)3(solv)3]+ with different thioether ligands leads to complexes Re1-Re5 and Re7 of the general formula [ReBr(CO3)L2] (Scheme 2). The compounds precipitated from the reaction mixtures as white powders and elemental analyses are in good agreement with the proposed formulations. As can be seen from Scheme 2, in methanol the functional groups do not participate in the coordination.

Table 2. Selected Bond Length (Å) and Angles (deg) of Complexes Re2, Re6, and Re8 Re2

Re6

Re8a

Re-C1 1.92(2) Re-C2 1.903(13) Re-C3 1.903(13) Re-S12 2.474(3) Re-S15 2.463(3) Re-Br 2.641(2)

Re-C(1) 1.932(8) Re-C(2) 1.914(7) Re-C(3) 1.912(7) Re-S(15) 2.458(2) Re-S(18) 2.507(2) Re-O(11) 2.149(5)

Re-C1 1.889(10) Re-C2 1.942(10) Re-C3 1.922(10) Re-S14 2.456(2) Re-S17 2.483(2) Re-O11 2.193(5)

C1-Re-C2 89.3(6) C1-Re-C3 90.0(6) C1-Re-S12 174.7(4) C1-Re-S15 90.7(5) C1-Re-Br 95.9(4) C2-Re-C3 91.2(5) C2-Re-S12 95.2(4) C2-Re-S15 179.4(4) C2-Re-Br 86.5(4) C3-Re-S12 92.7(4) C3-Re-S15 89.4(4) C3-Re-Br 173.7(4) S12-Re-S15 84.69(11) S12-Re-Br 81.61(9) S15-Re-Br 92.89(8)

C(3)-Re-C(2) 90.1(3) C(3)-Re-C(1) 88.5(3) C(2)-Re-C(1) 89.6(3) C(3)-Re-O(11) 96.7(2) C(2)-Re-O(11) 171.9(2) C(1)-Re-O(11) 94.9(2) C(3)-Re-S(15) 92.1(2) C(2)-Re-S(15) 95.0(2) C(1)-Re-S(15) 175.4(2) O(11)-Re-S(15) 80.5(13) C(3)-Re-S(18) 175.7(2) C(2)-Re-S(18) 93.2(2) C(1)-Re-S(18) 94.3(2) O(11)-Re-S(18) 79.8(13) S(15)-Re-S(18) 84.79(5)

C1-Re-C2 88.6(4) C1-Re-C3 88.3(4) C1-Re-S14 97.8(3) C1-Re-S17 95.5(2) C1-Re-O11 176.4(3) C2-Re-C3 89.9(4) C2-Re-S14 173.4(3) C2-Re-S17 93.4(2) C2-Re-O11 94.7(3) C3-Re-S14 91.8(3) C3-Re-S17 175.1(3) C6-Re-O21 93.5(3) S24-Re-S27 84.97(7) S24-Re-O21 78.7(2) S27-Re-O21 84.3(2)

a Two crystallographically independent molecules are contained in the asymmetric unit of the triclinic unit cell. Since there are no significant differences in the observed bond lengths and angles, only the values of one arbitrarily chosen individuum is discussed



Figure 2. Moleculare structure of complex Re8.


As a prototypic representative of this type of Re compound, complex Re2 was studied by X-ray diffraction analysis. Selected bond lengths and angles are listed in Table 2. As illustrated in Figure 1, the molecular structure of Re2 exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The Re-C distances [Re-C(1) ) 1.92 Å, Re-C(2) ) 1.90 Å, Re-C(3) ) 1.90 Å] are found in the range normal for this type of complexes (1.89-1.91 Å) (29, 52). Likewise, the Re-Br bond length is normal with 2.64 Å. The Re-S distances of 2.46 and 2.47 Å are comparable with other thioethercontaining rhenium complexes (54, 55). An alternative coordination of the C-C triple bond (53, 54) is not observed. A rather unexpected coordination behavior of the potentially tetradentate ligand 3,6-dithia-1,6-octanediol (L5) was observed with the trinitrato precursor complex [Re(NO3)3(CO)3]2-. It is available by treatment of (NEt4)2[ReBr3(CO)3] with an aqueous AgNO3 solution and subsequent precipitation of the halogen atoms as AgBr (see Scheme 3). Here, the ligand exchange reaction of L5 with [Re(NO3)3(CO)3]2- in THF led to the cationic complex Re8, showing the coordination of one hydroxy group. Scheme 3

Re8 of the formula [Re(CO)3(OHSSOH)]NO3 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction. The infrared spectrum of Re8 is very similar to those of complexes Re1 and Re2 but has an additional band at 1384 cm-1, indicating the N-O vibration of the nitrate counterion. Capillary electrophoretic investigations in aqueous solution confirm the cationic charge of Re8 in neutral solution. In alkaline medium (pH > 9), the hydroxy group deprotonates resulting in the formation of a neutral species of the proposed structure Re9. The molecular structure of Re8 shows the coordination of a hydroxy group at the rhenium center (Figure 2). Despite considering the lengthening of the Re-O(11) bond by the trans influence of the CO ligand, the distance of 2.19 Å is too long to be an alcoholato coordination, which is expected to be between 1.91 and 1.95 Å for a chelating OSS ligand in an oxorhenium(V) complex (55). With 2.161 Å, it is comparable with the ethanol coordination observed in (NEt4)[Re(NO)Br4(EtOH)] (56). All the other distances are similar to those found in complex Re1 described above (Table 2). Ligand exchange reaction of the carboxylic group bearing ligands L3 and L4 with (NEt4)2[ReBr3(CO)3] in water and with equimolar amounts of NaOH led to the complexes Re5 and Re6 in which the original bromide is replaced by the carboxylic group. The compounds precipitate from water and are practically unsoluble in all common organic solvents except DMSO. The elemental analyses show the absence of bromine. In the infrared spectra the absorption of the coordinating carbonyls is positioned at 2032, 1930, and 1900 cm-1. The vibration band of the carboxylic group is found at 1632 cm-1, i.e., 55 cm-1 lower than for the CO moiety of the ligands. This indicates the coordination of the deprotonated carboxylic group at the metal center completing the octahedral structure. The X-ray structure analysis of complex Re6 confirms this interpretation. As illustrated in Figure 3, the rhenium atom is centered in a distorted octahedron with facial coordination of the carbonyl ligands. The oxygen of one of the terminal carboxylic groups coordinates in trans position to the third carbonyl ligand. The second carboxylic group remains noncoordinated and offers an ideal site for functionalization or coupling a biomolecule. Technetium-99m Chemistry. No-Carrier-Added Preparation of the 99mTc Complexes. The no-carrier-added


Pietzsch et al. Table 3. HPLC Data of Re and 99mTc Complexes; Hypersil ODS: MeOH/0.01 M PBS pH 7.4, Linear Gradient 50 to 100 % A in 5 min


preparation of the neutral 99mTc(I) carbonyl thioether complexes Tc1-Tc4 could be performed with yields up to 90% using optimized preparation conditions (Scheme 4). To establish the structure of the 99mTc complexes prepared at tracer level, comparison by HPLC with the respective rhenium complexes prepared in macroscopic amounts was pursued applying parallel radiometric and photometric detection. Thus, after coinjection of complexes Tc1 and Re1, practically identical retention times were observed, while the recovery through the column was quantitative. Similarly, coinjections of the complex couples Tc2/Re2Tc6/Re6 led to identical Rt values for the respective compounds, revealing their structural analogy. Rt values of HPLC and partition coefficients (P) in the system octanol/saline are given in Table 3. As described above for the analogous rhenium complexes Re3-Re6, ligand exchange reaction of the carboxylic group bearing ligands L3 and L4 with [99mTc(H2O)3(CO)3]+ led to the formation of two 99mTc complex species in dependence on the reaction conditions. In acidic aqueous solution, complexes Tc3 and Tc4 containing a noncoordinated carboxylic group were formed. After neutralization of the reaction mixtures, ligand exchange reactions could be observed leading to the species Tc5 and Tc6 both with a coordinated carboxylic group. In contrast to the tridentate-coordinated compounds Tc5 and Tc6, which are stable during HPLC separation, ligand exchange reaction of the chlorine containing Scheme 4

complex

Rt (min)

Re1 Tc1 Re2 Tc2 Re3 Tc3 *Re4 *Tc4 Re5 Tc5 *Re6 *Tc6

7.6 7.7 6.7 6.7 3.3 3.2 10.5 10.6 6.5 6.5 11.4 11.5

log P (octanol/saline) 0.86 0.75 nd nd 0.41 -1.8

* For complex couples Tc4/Re4 and Tc6/Re6, the following gradient was used: (1) 5 min 0% A; (2) linear gradient 0 to 100% A in 5 min.

species Tc3 and Tc4 has to be suppressed by adding a saturated NaCl solution to the HPLC eluates. Stability of the Chlorine Containing 99mTc Complex Tc1 in Aqueous Solution. The behavior of the chlorine containing 99mTc complex Tc1 in aqueous neutral solutions such as 0.1 M phosphate buffer and saline at physiological pH value was investigated. Here, Tc1 serves as prototypical representative of Tc carbonyl complexes with bidentately coordinated thioethers without additional donor atom in the chelating unit. In saline, the chromatographically separated compound is stable for at least 120 min. However, in chloridefree aqueous solution, a water-coordinated cationic species of the proposed composition [99mTc(H2O)(CO)3L1]+ Tc1a occurs (Scheme 5). Thus, for biodistribution and plasma-binding studies, the complexes were used in a solution of 0.9% sodium chloride containing 25% propylene glycol for better solubility of the lipophilic products. Since Tc1a was not eluable from the Hypersil column, a Supelguard column with the elution conditions of protein binding studies was used (see Experimental Section). So, it was possible to eluate also the cationic complex Tc1a and to observe the time course of the conversion (Figure 4). The cationic charge of the conversion product was confirmed by paper electrophoresis (Figure 5) as well as by capillary electrophoresis. The ligand-exchange reaction leading to the cationic complex Tc1a proved to be reversible. After complete



Figure 4. Time course of conversion of the complex Tc1 into the complex Tc1a in a solution of 45% H2O/45%MeOH/10% propylene glycol.

Figure 5. Mobility of Tc1a in the electrical field (paper electrophoresis). Scheme 5

turnover of the parent compound into the cationic species, the parent compound can be reconstituted by an excess of sodium chloride (Figure 6). Obviously, the ligand exchange occurs only on the position of the chlorine atom, and the cationic species is the complex which does exist in an aqueous solution without excess of chloride ions. In phosphate buffer, the conversion of complex Tc1 is reversible, too, and the parent compound can be reconstituted by an excess of sodium chloride. The reversibility of the reaction was also confirmed by paper electrophoresis. Obviously, in phosphate buffer, the chlorine atom can be replaced not only by the water molecule but also by the phosphate ions of the buffer (Figure 7). Stability of the Carboxylic Group Bearing 99mTc Complexes Tc3-Tc6 in Aqueous Solution. The strong tendency of the carboxylic groups to coordinate the technetium core gives rise to conversion of the initially formed complexes Tc3 and Tc4, which still have the carboxylic group noncoordinated. So, these complexes were converted into the compounds Tc5 and Tc6 immediately after HPLC purification. Even in saturated solution of sodium chloride at pH 2.0, this conversion cannot be suppressed completely. It was not possible to obtain the Tc3 and Tc4 completely pure and stable for a longer period of time, and therefore, no biodistribution and plasma binding studies were performed. In contrast, Tc5 and Tc6 with a coordinated carboxylic group were stable in all investigated solutions at least for 120 min. Capillary electrophoretical studies of the complex couple Tc3/Tc5 support the assumption that the chloride ligand in the above described complexes of the general formula [99mTcCl(CO)3L] is responsible for their

Figure 6. HPLC pattern (radioactivity trace) of complex Tc1 (Supelguard column). (A) After 120 min incubation in water; (B) parent compound Tc1 reconstituted with NaCl after incubation in water for 120 min.

Figure 7. HPLC pattern (radioactivity trace) of complex Tc1 (Hypersil column). (A) After 120 min incubation in phosphate buffer 0.1 M, pH 7.4; (B) parent compound Tc1 reconstituted with NaCl after incubation in phosphate buffer for 120 min. Table 4. HPCE Migration Times of and Tc5 at Various pH values

pH 2.5 pH 7.0 pH 9.3

99mTc

Complexes Tc3

acetone mt (min)

complex Tc3 mt (min)

complex Tc5 mt (min)

8.4 8.5 5.3

8.5 9.7 6.1

8.3 8.5 5.5

reactivity in aqueous solution (Table 4). Tc3 was found to be an anionic species in the pH range 9.3-7.0 and a neutral one at pH 2.5, indicating the protonation of the free carboxylic group. Tc5 is a neutral species over the

422 Bioconjugate Chem., Vol. 11, No. 3, 2000 Table 5. HPLC Data of

99mTc

Pietzsch et al.

Tricarbonyl Complexes Incubated in Rat Plasma 30 min at 37 °Ca

complex

Supelguard Rt (min) of the parent compound

% protein bound activity after 30 min incubation in rat plasma (n ) 2)

% parent compound after 30 min incubation in rat plasma (n ) 2)

Tc1 Tc1a Tc2

4.4 0.5 2.9

71 ( 2 94 ( 6 70 ( 5

5(2 6(6 7(2

complex

Supelguard Rt (min) of the parent compound

% protein bound activity after 30 min incubation in rat plasma (n ) 2)

% parent compound after 30 min incubation in rat plasma (n ) 2)

Tc5 Tc6

0.5 0.8

56 ( 2 31 ( 2

44 ( 2 69 ( 2

a Supelguard: linear gradient 95% A to 40% A in 15 min [A, isopropanol/0.1% trifluoroacetic acid (TFA) (10/90); B, isopropanol/0.1% TFA (90/10)].

Table 6. Biodistribution Data of Complexes Tc1, Tc2, Tc5, and Tc6 in Male Wistar Rats complex

time p.i. min

blood % ID/g

brain

heart

lungs % ID

kidneys

liver

Tc1

5 120 5 120 5 120 5 120

1.01 ( 0.30 0.71 ( 0.14 0.92 ( 0.03 0.63 ( 0.06 1.09 ( 0.25 0.62 ( 0.06 0.91 ( 0.07 0.25 ( 0.02

1.03 ( 0.25 0.55 ( 0.08 0.78 ( 0.08 0.55 ( 0.07 0.07 ( 0.01 0.05 ( 0.01 0.09 ( 0.01

Chemical and Biological Characterization of Technetium(I

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