136
Bioconjugate Chem. 2003, 14, 136−143
Synthesis, Characterization, and Biological Evaluation of Technetium(III) Complexes with Tridentate/Bidentate S,E,S/P,S Coordination (E ) O, N(CH3), S): A Novel Approach to Robust Technetium Chelates Suitable for Linking the Metal to Biomolecules Hans-Juergen Pietzsch,*,† Sepp Seifert,† Rosemarie Syhre,† Francesco Tisato,‡ Fiorenzo Refosco,‡ Peter Leibnitz,§ and Hartmut Spies† Forschungszentrum Rossendorf, Institut fu¨r Bioanorganische und Radiopharmazeutische Chemie, PF 510119, D-01314 Dresden, Germany, Istituto di Chimica Inorganica e delle Superfici, Consiglio Nazionale delle Ricerche, Corso Stati Uniti 4, 35127 Padova, Italy, and Bundesanstalt fu¨r Materialforschung, Richard-Willsta¨tter-Str. 11, D-12489 Berlin, Germany. Received July 16, 2002; Revised Manuscript Received November 13, 2002
A novel type of mixed-ligand Tc(III) complexes, [Tc(SCH2CH2-E-CH2CH2S)(PR2S)] (E ) S, N(CH3); PR2S ) phosphinothiolate with R ) aryl, alkyl) is described. These “3+2”-coordinated complexes can be prepared in a two-step reduction/substitution procedure via the appropriate chloro-containing oxotechnetium(V) complex [TcO(SES)Cl] {E ) S, N(CH3)}. Tc(III) compounds have been fully characterized both in solid and solution states and found to adopt the trigonal-bipyramidal coordination geometry. The equatorial trigonal plane is formed by three thiolate sulfur atoms, whereas the phosphorus of the bidentate P,S ligand and the neutral donor of the tridentate chelator occupy the apical positions. The 99Tc(III) complexes have been proven to be identical with the 99mTc agents prepared at the no-carrier-added level by comparison of the corresponding UV/vis and radiometric HPLC profiles. Challenge experiments with glutathione clearly indicate that this tripeptide has no effect on the stability of the 99mTc complexes in solutions. Biodistribution studies have been carried out in rats at 5 and 120 min postinjection. The substituents at the bidentate P,S ligand significantly influence the biodistribution pattern. Remarkable differences are observed especially in brain, blood, lungs, and liver. All the complexes are able to penetrate the blood-brain barrier of rats and showed a relatively fast washout from the brain.
INTRODUCTION
This paper reports on the synthesis and biological characterization of a novel class of technetium(III) mixedligand complexes with tridentate S,E,S and bidentate P,S coordination (E ) S, N(R), O). The syntheses of these mixed-ligand species represent an alternative approach for the development of radiotracers because they offer manifold possibilities to combine the metal with a biologically active fragment and to vary bio-relevant molecular properties. Mixed-ligand complexes of technetium and rhenium contain the metal ion stabilized in an appropriate oxidation state by a suitable ligand framework, which only partially fills the coordination sphere. Examples for such arrangements are described, beside the technetium(I) and rhenium(I) tricarbonyl moiety [M(CO)3]+ (1), mainly for Tc(V). These include the “super-nitrido” fragment [Tc(N)(PNP)]2+ (PNP ) tridentate aminediphosphine ligand) (2), the substitution-inert [Re(O)(L)]2+ moiety (L ) bidentate functionalized phosphine) (3), and the [M(O)(SES)]+ group (SES ) tridentate dithiol ligand; E ) S, NR, O) forming the well-known “3+1” Tc and Re complexes (4, 5). In all cases the coordination sphere of the metal ion is completed by additional mono-, bi-, or * Corresponding author. Fax: (0351) 260 3232. E-mail:
[email protected]. † Institut fu ¨ r Bioanorganische und Radiopharmazeutische Chemie. ‡ Istituto di Chimica Inorganica e delle Superfici. § Bundesanstalt fu ¨ r Materialforschung.
tridentate co-ligands. Among these species, the so-called “3+1” complexes suffer in vitro and in vivo substitution reaction with thiol-containing molecules such as cysteine or glutathione (6-9). To overcome this problem, labile oxo-Tc(V) compounds have been replaced by electron-rich Tc(III) ones in the search for neutral and stable mixedligand complexes. So, “4+1” Re and Tc complexes containing 2,2′,2′′-nitrilotris-(ethanethiol) as a tripodal ligand and tertiary phosphines or isocyanides as co-ligands have shown superior properties compared to the “3+1” complexes in terms of stability (10-12). Another type of neutral Tc(III) complexes derived from the reaction of oxo-Tc(V) “3+1“ precursors with tertiary phosphines, namely, the “3+1+1” complexes of general formula [M(PR3)(SES)(SR)] (SES ) tridentate dithiol ligand; E ) S, NR, O), are still unstable against cysteine and GSH and, sometimes, re-oxidize to the original starting material (13, 14). Stability of this class of compounds can be enhanced by combining the monodentate ligands, i.e., tertiary phosphine and thiol, into a bidentate P,S phosphinothiol to produce “3+2” Tc(III) mixed-ligand complexes of the type [Tc(SES)(R2PS)]. Previous investigations have demonstrated that P,S functionalized phosphines allow the synthesis of Tc(III) species of different geometry by varying either the nature of the carbon chain between the phosphorus and the sulfur atoms and/or the substituents at the phosphorus atom (15, 16). Furthermore bis- and monosubstituted nitrido-Tc(V) complexes with phosphinothiolate ligands have been described (17).
10.1021/bc025575v CCC: $25.00 © 2003 American Chemical Society Published on Web 12/05/2002
Technetium(III) Complexes
The aim of the work presented here was to prepare neutral and stable “3+2” Tc(III) complexes both at macroscopic and at nca level, to confirm their identity by using chromatographic techniques coupled with UV/ vis and radiometric detection, to study their reactivity in various media and to check the biodistribution properties in rats of a number of model complexes. EXPERIMENTAL PROCEDURES
General. All solvents and commercially available substances were of reagent grade and used without further purification. The P,S ligands were prepared by ARGUS Chemicals according to the method reported by Chatt et al. (18). The tridentate ligands 3-thiapentane1,5-dithiol and 3-oxapentane-1,5-dithiol were obtained from Aldrich. 3-(N-Methyl)azapentane-1,5-dithiol was prepared according to standard procedures starting from N-methyl-2,2′-iminodiethanol (MERCK-Schuchart) (19). 99 Tc as NH4TcO4 was obtained from Amersham as 0.3 M aqueous solution.99mTcO4- was eluated from a commercial 99Mo/99mTc generator (Mallinckrodt). Instrumentation. 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 Perkin-Elmer FTIR-spectrometer SPECTRUM 2000. UV/vis spectra were recorded on a SPECORD S10 spectrometer from Carl Zeiss Jena. Proton, 13C, and 31P NMR spectra were collected on a Bruker 300 instrument, using SiMe4 as internal reference (1H and 13C) and 85% aqueous H3PO4 as external reference (31P). Samples were dissolved in deuterated chloroform at a concentration of ca. 2%. Chemical shifts are reported as δ in ppm. Cyclic voltammetry of complexes 1-5 was carried out in dichloromethane solutions (3.5 × 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 T ) 293 K. Potentials were measured relative to an Ag-wire pseudoelectrode using the Fc/Fc+ couple as internal reference. Controlled potential coulometries of dichloromethane solutions of 1 were performed using an Amel model 721 integrator, in a H-shaped cell containing, in arm 1, a platinum-gauze 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. Thinlayer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses were performed for controlling the identity, the radiochemical purity, and the stability of the preparations. For TLC studies silica gel plates (Merck 60F254) and mobile phases of MeOH/0.1 N HCl (95/5), acetone, or BuOH/water/MeOH/25% ammonia (60/20/20/1) were used. The plates were scanned with a Raytest Rita radioanalyzer. HPLC analyses were performed on a Perkin-Elmer device consisting of a Turbo LC System with a quaternary pump (Series 200 LC Pump), a Programmable Absorbance Detector Model 785A, and a homemade γ-ray detector (Bohrloch, NaI(Tl) crystal). HPLC analyses were carried out with a Hypersil ODS column (250 × 4 mm) using a gradient eluent of acetonitrile/0.05 M NH4Ac pH 4, and a flow rate of 1.0 mL/min. The effluent from the column was monitored by UV absorbance at 254 nm for 99Tc reference complexes or γ-ray detection for the 99mTc complexes. 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
Bioconjugate Chem., Vol. 14, No. 1, 2003 137 Table 1. Crystal Data and Structure Refinement for Complexes [99Tc]1 and [99Tc]4 complex formula f.w. crystal system space group a [Å] b [Å] c [Å] R [deg] β [deg] γ [deg] V [Å3] Z temp [K] d [g/cm3] abs. coeff. [mm-1] F(000) µ [Å] radiation crystal size [mm3] 2θ range hkl no. of coll. reflns no. of indep. reflns. GOF R [I > 2s (I)] R (all data) largest diff. peak largest diff. hole
[99Tc]1 C18H22PS4Tc 495.57 triclinic P-1 7.11(2) 17.00(4) 19.18(5) 64.06(8) 88.60(9) 87.95(9) 2084(9) 4 293(2) 1.579 1.167 1008 0.71073 0.540 × 0.072 × 0.036 2.13-28.84 -9 e h e 9 -22 e k e 17 -18 e l e 24 6175 6154 1.195 R1 ) 0.0814 wR2 ) 0.1779 R1 ) 0.1015 wR2 ) 0.2017 1.875 -1.019
[99Tc]4 C19H25NPS3Tc 492.55 monoclinic P2(1)/n 14.8299(7) 8.0082(4) 17.7414(9) 90 97.0470(10) 90 2091.1(2) 4 293(2) 1.565 1.067 1008 0.71073 0.9 × 0.54 × 0.18 1.69-28.91 -20 e h e 11 -10 e k e 10 -23 e l e 23 12200 5029 1.093 R1 ) 0.0372 wR2 ) 0.1025 R1 ) 0.0399 wR2 ) 0.1048 0.779 -0.736
monochromatized Mo-KR radiation (λ ) 0.71073 Å). A summary of the crystallographic data is given in Table 1. The positions of the non-hydrogen atoms were determined by the heavy atom technique. After anisotropic refinement of these positions, 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. Relevant bond lengths and angles are contained in Table 3. Atomic positional and thermal parameters, full lists of bond lengths and angles, and Fo/Fc values have been deposited (20). Synthesis of [TcO(SCH2CH2SCH2CH2S)Cl] (I) and [TcO(SCH2CH2N(CH3)CH2CH2S)Cl] (II). These precursor complexes were prepared by ligand-exchange reaction of [N(C4H9)][TcOCl4] with the appropriate tridentate ligand as described elsewhere (21, 22). Synthesis Procedure for the Tc(III) Complexes [Tc(SCH2CH2SCH2CH2S)(PR2S)]-[99Tc]1-[99Tc]3. A 100 µmol sample of the oxotechnetium(V) complex I and 400 µmol of the appropriate phosphinethiol were dissolved in 3 mL of acetonitrile. After addition of 0.5 mL of acetic acid, the mixture was stirred at room temperature under argon for 60 min. The color of the solution turned from yellowish-brown to violet. The reaction mixture was reduced in volume to 1 mL, and methanol was added until the solution became turbid. The mixture was then allowed to stand overnight in the refrigerator. Recrystallization of the raw precipitates from chloroform/ methanol at -20 °C gave [99Tc]1, [99Tc]2, and [99Tc]3 as dark-violet crystals. [Tc(SCH2CH2SCH2CH2S)(PPh2S)]-[99Tc]1. Yield (rel. to I): 66%. mp ) 175 °C. Anal. (C18H22PS4Tc) Calcd: C 43.5%, H 4.5%, S 25.8%. Found: C 43.3%, H 4.8%, S 25.3%. UV/vis (CHCl3): λmax(lg) ) 286 nm (4.0), 358 (3.6), 538 (2.7). IR (KBr): νC-H ) 2912-3070 cm-1, νCdC(aromat) ) 1582, 1619 cm-1, νP-C ) 1433 cm-1.1H NMR: 2.49 (m,
138 Bioconjugate Chem., Vol. 14, No. 1, 2003 Table 2. Selected Data for
99Tc(III)
Pietzsch et al.
Complexes
ligand set SES
R in PR2S
Tc-Pa
Tc-Ea,b
31P{1H} c,d
E° e TcIII/TcII
Epa e TcIII/TcIV
[99Tc]1 [99Tc]2 [99Tc]3 [99Tc]4 [99Tc]5
SSS SSS SSS SNS SNS
Ph Cy Me Ph Me
2.320
2.402
2.298
2.248
81.5 (+99.0) 87.8 (+92.0) 56.6 (+107.8) 88.5 (+106.0) 62.8 (+114.0)
-0.468 -0.550 -0.565 -0.556 -0.568
0.294 0.200 0.215 0.144 0.247
[99Tc] “3+1+1” (13) [99Tc]“3+1+1” (13)
SSS SNS
Me2Ph/S Me2Ph/S
2.358 2.318
2.398 2.273
20.4 (+65.2) 31.4 (+76.2)
-0.335 -0.520
0.262 0.063
complex
a In Å. b E is the central atom of the tridentate ligand. c In ppm. d The chemical shift between uncoordinated and coordinated phosphinothiol or phosphine is reported in parentheses. e In mV, potentials are vs the ferricinium/ferrocene couple.
Table 3. Selected Bond Lengths and Angles of Complexes [99Tc]1 and [99Tc]4 [99Tc]1 Tc-S(1) Tc-S(2) Tc-S(3) Tc-S(4) Tc-P(1) S(2)-Tc-S(3) S(2)-Tc-S(1) S(3)-Tc-S(1) S(2)-Tc-P(1) S(3)-Tc-P(1) S(1)-Tc-P(1) S(2)-Tc-S(4) S(3)-Tc-S(4) S(1)-Tc-S(4) P(1)-Tc-S(4)
[99Tc]4 2.251(6) 2.216(6) 2.239(7) 2.402(7) 2.320(7) 114.9(2) 118.5(2) 126.4(2) 93.8(2) 94.4(2) 85.2(2) 87.9(2) 87.0(2) 91.9(2) 177.04(10)
Tc(1)-S(3) Tc(1)-S(1) Tc(1)-S(2) Tc(1)-N(1) Tc(1)-P(1) S(3)-Tc(1)-S(1) S(3)-Tc(1)-S(2) S(1)-Tc(1)-S(2) S(3)-Tc(1)-N(1) S(1)-Tc(1)-N(1) S(2)-Tc(1)-N(1) S(3)-Tc(1)-P(1) S(1)-Tc(1)-P(1) S(2)-Tc(1)-P(1) N(1)-Tc(1)-P(1)
2.2202(9) 2.2396(8) 2.2427(8) 2.248(2) 2.2979(7) 121.27(3) 116.57(3) 121.73(3) 85.30(8) 92.64(8) 85.30(8) 92.29(3) 85.28(3) 99.27(3) 175.42(8)
4H), 2.96 (m, 8H), 7.35-7.60 (10H, PPh2). 13C NMR: 34.99 (s), 35.72 (d), 36.53 (d), 38.75 (d), 128.14 (d), 129.74 (s), 132.73 (d), 135.48 (d). 31P NMR: 81.48 (bs). [Tc(SCH2CH2SCH2CH2S)(P(C6H11)2S)]-[99Tc]2. Yield (rel. to I): 63% rel. to I. mp ) 158 °C. Anal. (C18H34PS4Tc) Calcd: C 42.5%, H 6.7%, S 25.2%. Found: C 42.4%, H 6.8%, S 24.7%. UV/vis (CHCl3): λmax(lg) ) 285 nm (4.0), 356 (3.6), 552 (2.8). IR (KBr): νC-H ) 2910-3075 cm-1, νCdC(aromat) ) 1582, 1615 cm-1, νP-C ) 1436 cm-1. 1H NMR: 1.59 (d, 6H; P-CH3), 1.84 (dt, 2H; P-CH2-), 2.89 (m, 8H), 3.03 (s, 3H; N-CH3), 3.25 (m, 2H). 13C NMR: 17.82 (d), 36.10 (d), 37.83 (d), 39.44 (d), 49.99 (s), 61.57 (s). 31P NMR: 62.80 (bs). [Tc(SCH2CH2SCH2CH2S)(PMe2S)]-[99Tc]3. Yield (rel. to I): 71%. mp ) 162 °C. Anal. (C8H18PS4Tc) Calcd: C 25.8%, H 4.9%, S 34.4%. Found: C 25.7%, H 4.9%, S 34.0%. UV/vis (CHCl3): λmax(lg) ) 283 nm (4.0), 355 (3.6), 548 (2.7). IR (KBr): νC-H ) 2850-3065 cm-1, νP-C ) 1435 cm-1. 1H NMR: 1.25-2.35 (20H + 2H), 2.43 (m, 2H), 2.93 (m, 8H). 13C NMR: 26.0-29.3 (Ccyclohexyl), 34.53 (d), 37.48 (d), 39.02 (d), 40.10 (d). 31P NMR: 87.80 (bs). Synthesis Procedure for the Tc(III) Complexes [Tc(SCH 2 CH 2 N(CH 3 )CH 2 CH 2 S)(PR 2 S)]-[ 99 Tc]4,[99Tc]5. A 100 µmol sample of the oxotechnetium(V) complex II and 400 µmol of the appropriate phosphinethiol were dissolved in 3 mL of acetonitrile. After addition of 0.5 mL of acetic acid, the mixture was stirred at room temperature under argon for 90 min. The color of the solution turned from yellowish-brown to violet. After reduction of the reaction mixture in volume to 0.5 mL, the complexes were isolated by column chromatography (column 10 × 250 mm, stationary phase silica gel 0.04-0.063 mm, eluent CHCl3/acetone 12:1v/v). Recrystallization from chloroform/methanol (1:3 v/v) gave violet crystals of [99Tc]4 and [99Tc]5. [Tc(SCH2CH2N(CH3)CH2CH2S)(PPh2S)]-[99Tc]4. Yield (rel. to II): 68%. mp ) 185 °C. Anal. (C19H25NPS3Tc)
Calcd: C 46.2%, H 5.1%, N 2.8%, S 19.5%. Found: C 46.6%, H 5.5%, N 2.8%, S 18.9%. UV/vis (CHCl3): λmax(lg) ) 280 nm (4.2), 349 (3.7), 545 (2.9). IR (KBr): νC-H ) 2915-3070 cm-1, νCdC(aromat) ) 1580, 1623 cm-1, νP-C ) 1433 cm-1. 1H NMR: 2.39 (q, 2H), 2.85 (m, 4H), 2.99 (m, 4H), 3.11 (s, 3H; N-CH3), 3.23 (m, 2H), 7.35-7.55 (10H, PPh2). 13C NMR: 35.01 (d), 37.29 (d), 39.55 (d), 49.95 (s), 61.78 (s), 127.90 (d), 129.27 (s), 132.48 (d), 137.21 (d). 31P NMR: 88.50 (bs). [Tc(SCH2CH2N(CH3)CH2CH2S)(PMe2S)]-[99Tc]5. Yield (rel. to II): 64%. mp ) 167 °C. Anal. (C9H21NPS3Tc) Calcd: C 29.3%, H 5.7%, N 3.8%, S 26.1%. Found: C 29.6%, H 5.8%, N 3.5%, S 25.8%. UV/vis (CHCl3): λmax(lg) ) 276 nm (4.3), 345 (3.8), 509 (2.8). IR (KBr): νC-H ) 2860-3069 cm-1, νP-C ) 1434 cm-1. 1H NMR: 1.59 (d, 6H; P-CH3), 1.84 (dt, 2H; P-CH2-), 2.89 (m, 8H), 3.03 (s, 3H; N-CH3), 3.25 (m, 2H). 13C NMR: 17.82 (d), 36.10 (d), 37.83 (d), 39.44 (d), 49.99 (s), 61.57 (s). 31P NMR: 62.80 (bs). General Synthesis Procedure for the No-CarrierAdded Preparation of the Compounds [99mTc(SES)(PR2S)]-[99mTc]1, [ 99mTc]2, [99mTc]4, [99mTc]6-[99mTc]11. No-carrier-added preparations of “3+2” Tc(III) compounds were studied with the tridentate SES ligands 3-thiapentane-1,5-dithiol, 3-(N-methyl)azapentane-1,5dithiol and 3-oxapentane-1,5-dithiol and bidentate P,S ligands illustrated in Figure 4. A 0.10 mg (for preparing [Tc(SSS/PR2S)]) or 0.05 mg (for preparing [Tc(SNMeS/PR2S)] and [Tc(SOS/PR2S)]) sample of the bidentate P,S ligand, dissolved in 0.10 mL of ethanol, and 0.05 mg of the appropriate tridentate SES ligand, dissolved in 0.10 mL of ethanol, were added to a mixture of 0.50-1.0 mL pertechnetate, 0.25 mL propylene glycol, and 0.75 mL of acetonitrile. After addition of 40 µl 0.1 N NaOH, the complexes were formed by reduction of pertechnetate with 40 µl SnCl2 solution (2.0 mg SnCl2 dissolved in 5.0 mL 0.1 N HCl). The reaction was completed by heating at 50 °C within 15 min (yields between 60 and 90%). The preparations were purified for stability and biodistribution studies by HPLC using a semipreparative Hypersil column (250 × 8 mm, 10 µm). Determination of Partition Coefficients. Partition coefficients of the 99mTc complexes were determined by shake-flask method by mixing 0.5 mL of the complex solution (0.9% NaCl solution and 20% propylene glycol) and 0.5 mL n-octanol and shaking for 5 min. After centrifugation aliquots of both phases were counted and the partition coefficient calculated. Ligand Exchange (Challenge) Experiments with Glutathione (GSH). 99mTc complexes, dissolved in a mixture of propylene glycol/MeOH (2:1 v/v), were diluted with an equivalent volume of an aqueous solution of 20 mM GSH containing 25% of propylene glycol immediately
Technetium(III) Complexes Scheme 1. Reaction Routes to Tc(III) Complexes with “3+2” Coordination (E ) N(CH3), S)
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 50 µL sample 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% to 40% A in 15 min [A, 2-propanol/0.1% trifluoroacetic acid (TFA) (10/90); B, 2-propanol/0.1% TFA (90/10)]. Biodistribution Studies. The animal studies in male Wistar rats (5-6 weeks old) were carried out according to the relevant national regulations. A 500 µL sample 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 at 5 and 120 min p.i.. Selected organs were isolated for weighing and counting. The accumulated radioactivity in the organs tissue were calculated in terms of percentage of injected dose per organ as well as percent injected dose per gram of blood. RESULTS AND DISCUSSION
Technetium-99 Chemistry. Synthesis. As shown in Scheme 1, the Tc(III) mixed-ligand complexes can be prepared in a two-step substitution-reduction procedure starting from the prereduced precursor [TcOCl4]- via the appropriate chloro-containing oxotechnetium(V) complex I or II. In this case, sequential addition of the relevant dithiol followed by the bidentate phosphinothiol is required. An alternative one-pot reaction with the simultaneous addition of the ligands to the labile [TcOCl4]precursor is possible, but the yield of the Tc(III) species is lower. In these reactions the phosphinethiols work as reducing agent as well as coordinating ligand. Both procedures are characterized by a typical change in color from orange to deep-violet indicating the transfer from Tc(V) to Tc(III). There is no evidence for the formation of other oxo-Tc(V) species. Characterization. The Tc(III) complexes have been characterized by (i) elemental analyses, which are in
Bioconjugate Chem., Vol. 14, No. 1, 2003 139
agreement with the proposed formulations, (ii) cyclic voltammetry, (iii) spectroscopic measurements, including IR, UV/vis, NMR, and (iv) X-ray structure analyses of two prototypic representatives. The IR spectra of all compounds show characteristic absorptions of the Tc-P stretching vibrations. No bands assignable to the TcdO core are observed in the range 900-1000 cm-1 indicating the absence of oxotechnetium species. The UV/vis spectra of the Tc(III) complexes exhibit very similar and characteristic intense bands at 345-353 nm and less intense absorptions in the visible region (509552 nm). The diamagnetism exhibited by this class of Tc(III) complexes is in agreement with low-spin d4 trigonal bipyramidal configuration (vide infra). Consequently, NMR spectra show sharp proton and carbon signals and allow to distinguish both coordinated dithiolate and phosphinothiolate fragments. On the contrary, roomtemperature 31P{1H} spectra exhibit rather broad signals due to the quadrupolar relaxation induced by the 99Tc nucleus (I ) 9/2) at the neighbor P atom (23). The 31P signal moves significantly downfield by ca. 100 ppm upon phosphine coordination (see Table 2) compared to the values exhibited by uncoordinated PMe2S ) - 51.2, PPh2S ) - 17.5, and PCy2S ) - 4.2 ppm. Despite the more shielded values exhibited by those complexes containing the more basic phosphine of the series (i.e., PMe2S), this ligand induces the highest chemical shift variation (+114 and +107.8 ppm for [99Tc]5 and [99Tc]3, respectively), indicating a strong contribution of the phosphine lone pair to the metal-phosphorus bond. Less pronounced is the chemical shift variation induced by the less basic PCy2S and PPh2S ligands (see Table 2). The nature of the trans-axial heteroatom of the tridentate ligand (S or N) affects the 31P signal and the Tc-P bond as well. In fact, by increasing the π-accepting combination in the series (thioether/phosphine in [99Tc]1 vs amine/ phosphine in [99Tc]4), the Tc-P bond distance elongates from 2.298 to 2.320 Å, whereas the 31P signal is less downfield shifted. These “3+2” species do not rearrange back to the original oxo-Tc(V) precursors, as it was observed in the case of some isostructural Tc(III) “3+1+1” complexes containing a monodentate phosphine and a monodentate thiolate (13). Cyclic voltammetric (CV) data support the observations pointed out above. CV oxidation (Epa) and reduction (E°) potentials are reported in Table 2 and a representative voltammogram is depicted in Figure 1. The redox stability window for this class of Tc(III) “3+2” complexes is about 700-800 mV. This range is ca. 100-200 mV larger when compared to the redox windows found in similar T(III) “3+1+1” complexes, indicating an increased stability of the more chelated “3+2” compounds. Complexes [99Tc]1 and [99Tc]4, as prototypic representatives of this novel class of neutral Tc(III) compounds, were studied by X-ray structure analysis. A summary of the crystallographic data is given in Table 1. Selected bond lengths and angles are cumulated in Table 3. As illustrated in Figures 2 and 3, the complexes adopt the trigonal-bipyramidal geometry comprised by three sulfur atoms, the neutral heteroatom of the tridentate ligand, and the tertiary phosphorus atom. The trigonal plane is formed by the three thiolate sulfurs of the tridentate ligand and the bidentate P,S chelator. The phosphorus and the neutral heteroatom of the tridentate chelate ligand occupy the apical positions. This arrange-
140 Bioconjugate Chem., Vol. 14, No. 1, 2003
Pietzsch et al.
Figure 3. Molecular structure of complex [99Tc]4. Figure 1. Cyclic voltammogram of complex [99Tc]5 in 1 mM CH2Cl2 solution. Potentials are vs Ag/Ag+. The scan rate is 200 mV s-1.
Scheme 2. No-Carrier-Added Preparation of “3+2” Coordinated 99mTc Complexes
Table 4. log Po/w Values and Reaction against Rat Plasma of “3+2” 99mTc Model Complexes
Figure 2. Molecular structure of complex [99Tc]1.
ment is quite common for transition-metal complexes containing two π-acceptor groups (12, 13, 15, 24-26). The Tc-Sthiolato distances are restricted in a narrow range (2.21-2.24 Å) and compares well with those previously reported for similar trigonal-bipyramidal complexes (12, 13, 26). The unique axial metal thioethersulfur bond in [99Tc]1 elongates to 2.427 Å. The P-Tc-E axis shows only slight deviation from linearity. Other angles of the inner coordination sphere are consistent with the tbp arrangement. A special order/disorder phenomenon was detected in the crystal packing of [99Tc]4. As frequently observed in this type of complexes, the ‘‘SNS′′ chelator can change its conformation by a flipflop mechanism giving rise to a statistical distribution of two equal isomers within the crystal (27). Technetium-99m Chemistry and Biodistribution Studies. No-Carrier-Added Preparation of the 99mTc Complexes. The “3+2” coordinated 99mTc complexes [99mTc]1, [99mTc]2, [99mTc]4, and [99mTc]6-[99mTc]11 were prepared by a one-step procedure starting from 99mTcO - with stannous chloride as reducing agent in 4 nearly neutral solutions using optimized amounts of the bidentate P,S ligands as well as of the tridentate S,E,S
complex
log Po/w
plasma binding
[99mTc]1 [99mTc]2 [99mTc]4 [99mTc]6 [99mTc]7 [99mTc]8 [99mTc]9 [99mTc]10 [99mTc]11
3.1 1.5 1.5 1.2 1.5 1.6 2.2 1.8 1.2
+ + +
ligands (Scheme 2). While 0.05 mg of the P,S chelators are sufficient for the preparation of the complexes with S,N,S/P,S and S,O,S/P,S coordination, the formation of the S,S,S/P,S complexes needs 1.0 mg of the P,S ligand. For all products the amount of the tridentate ligand was determined to be 0.05 mg. To establish the structure of the 99mTc complexes, comparison by HPLC with the well-characterized 99Tc analogues was pursued applying parallel radiometric and photometric detection. Thus, after co-injection of appropriate 99mTc/99Tc complex couples, practically identical retention times were observed, while the recovery through the column was quantitative. Examination of the octanol/water partition coefficients suggests high lipophilicity for the resulting “3+2” 99mTc species (Table 4).
Technetium(III) Complexes
Bioconjugate Chem., Vol. 14, No. 1, 2003 141
Figure 4. Plasma binding of [99mTc]9 and [99mTc]7 determined by HPLC. (A) [99mTc]9 after 5 min incubation at 37 °C; (B) after 150 min; (C) [99mTc]7 after 5 min. Dotted line, UV detection (220 nm); solid line, γ detection [99mTc]9 Table 5. Biodistribution of “3+2” Coordinated (%ID/g, 5 and 120 min p.i., mean ( SD, N ) 5)
99mTc
Complexes in the Blood and in Selected Organs of Wistar Rats %ID/g
complex
[min p.i.]
blood
brain
heart
lung
kidney
liver
[99mTc]1
5 120 5 120
0.3 ( 0.1 e0.1 0.6 ( 0.1 0.2 ( 0.05
0.40 ( 0.03
