Mixed-Ligand Technetium(III) Complexes with Tetradendate

538

Bioconjugate Chem. 2001, 12, 538−544

Mixed-Ligand Technetium(III) Complexes with Tetradendate/ Monodendate NS3/Isocyanide Coordination: A New Nonpolar Technetium Chelate System for the Design of Neutral and Lipophilic Complexes Stable in Vivo H.-J. Pietzsch,*,† A. Gupta,† R. Syhre,† P. Leibnitz,‡ and H. Spies† Forschungszentrum Rossendorf, Institut fu¨r Bioanorganische und Radiopharmazeutische Chemie, PF 510119, D-01314 Dresden, Germany, and Bundesanstalt fu¨r Materialforschung und -pru¨fung, Rudower Chaussee 5, Haus 8.15, D-12489 Berlin, Germany. Received November 15, 2000

Starting from the tripodal ligand 2,2′,2′′-nitrilotris(ethanethiol) (NS3) and isocyanides (CNR) as coligands, neutral mixed-ligand technetium(III) complexes of the general formulation [Tc(NS3)(CNR)] have been synthesized and characterized. The 99Tc complexes can be obtained by a two-step reduction/ substitution procedure starting from [TcO4]- via the phosphine-containing precursor complex [Tc(NS3)(PMe2Ph)]. As shown by X-ray structural analyses, the complexes adopt a nearly ideal trigonalbipyramidal geometry with the trigonal plane formed by the three thiolate sulfurs of the tripodal ligand. The central nitrogen atom of the chelate ligand and the monodendate isocyanides occupy the apical positions. The no-carrier-added preparation of the corresponding 99mTc complexes was performed by a one-step procedure starting from 99m[TcO4]- with stannous chloride as reducing agent. Biodistribution studies in the rat demonstrated for the nonpolar, lipophilic compounds a significant initial brain uptake. In vitro challenge experiments with glutathione clearly indicated that no transchelation reaction occurs. Furthermore, there were no indications for reoxidation of Tc(III) to Tc(V) species or pertechnetate. We propose this type of complexes as a useful tool in the design of lipophilic 99mTc or 186Re/188Re radiopharmaceuticals.

INTRODUCTION

Complexes of the γ-emitting nuclide technetium-99m find broad application in diagnostic nuclear medicine because of its optimal nuclide properties (1-4). To design new and broadly applicable “bioactive” Tc-99m radiopharmaceuticals able to be involved in metabolic processes or to bind to receptors, the radioactive metal has to be incorporated into biologically active molecules in form of chelates (5, 6). The most frequently used technetium compounds suitable for coupling the metal to biologically active molecules are square-pyramidal complexes of the oxo ion [TcdO]3+ based on tetradentate N2S2 ligands (7, 8). In this context, the “3 + 1” mixed-ligand approach is of high interest too (9-11). Properties and thus the in vivo behavior of such complexes are strongly influenced by the presence of the quite polar [TcdO]3+ unit. Whether such a polarity is beneficial or not is not obvious and depends on the requirements for specific radiotracers, e.g., for receptor targeting agents or for metabolic tracers. Another crucial point in tracer design is the stability of the respective Tc chelate toward ligand exchange in vivo. Recently, it was shown that the applicability of the “3 + 1” mixed-ligand Tc(V) chelates is limited because of their reaction with glutathione in vivo (12, 13). Thus, there is a considerable interest in alternative chelate systems that offer lower polarity and enhanced in vivo stability. Such systems are based on * To whom correspondence should be addressed. Fax: (0351) 260 3232. E-mail: [email protected]. † Forschungszentrum Rossendorf. ‡ Bundesanstalt fu ¨ r Materialforschung und -pru¨fung.

oxo-free cores with the metal at lower oxidation states. Thus, Tc(I) carbonyl complexes resulting from the reaction of the organometallic Tc(I) aqua ion [Tc(OH2)3(CO)3]+ as a precursor with either N-containing ligands, phosphines, or thioether ligands (14-19) are hopeful candidates for stable binding of Tc to peptides and other polar biomolecules. Here we offer a new type of Tc(III) chelate formed by the tripodal 2,2′,2′′-nitrilotris(ethanethiol) and a monodentate isocyanide that should fulfill the requirements for a lipophilic, nonpolar building block stable against ligand exchange reaction in vivo. Part of the family of “n + 1” mixed-ligand technetium and rhenium species (20-22), such a “4 + 1” chelate offers the advantage of high versatility in conjugating biomolecules as described for the “3 + 1” series (23). In the present paper, we report on the synthesis and structural analysis of 99Tc complexes with simple isocyanides serving as models for functionalized derivatives. The no-carrier-added preparation of the analogous 99mTc complexes, studies on stability in aqueous solution and in plasma, as well as biodistribution studies in rats are dealt with. EXPERIMENTAL PROCEDURES

General Methods. Dimethylphenylphosphine (PMe2Ph) was purchased from Aldrich. Isocyanides were obtained from Aldrich (benzyl isocyanide), Merck (cyclohexyl isocyanide), and Fluka (isocyanoacetic acid ethyl ester). All solvents and commercially available substances were of reagent grade and used without further purification. The tripodal ligand 2,2′,2′′-nitrilotris(ethane-

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Mixed-Ligand Technetium(III) Complexes

Bioconjugate Chem., Vol. 12, No. 4, 2001 539

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

99Tc1, 99Tc2, 99Tc3,

and

99Tc4

99Tc1

99Tc2

99Tc3

99Tc4

C14H23NPS3Tc 430.48 monoclinic P2(1)/c 13.3502(5) 9.4833(4) 15.3085(6) 90 112.7420(10) 90 1787.44(12) 4 293(2) 1.600 1.236 880 0.71073 0.9 × 0.324 × 0.072 1.65-23.31 -11 e h e 14 -10 e k e 10 -17 e l e 16 7757 2561 1.096 R1 ) 0.0218 wR2 ) 0.0578 R1 ) 0.0230 wR2 ) 0.0590

C14H19N2S3Tc 409.49 monoclinic P2(1)/c 8.2607(4) 11.1868(5) 17.6186(9) 90 93.8360(10) 90 1624.50(14) 4 293(2) 1.674 1.263 832 0.71073 0.90 × 0.72 × 0.36 2.16-28.98 -11 e h e 8 -14 e k e 11 -23 e l e 20 9450 3900 1.206 R1 ) 0.0982 wR2 ) 0.2441 R1 ) 0.1033 wR2 ) 0.2618

C13H23N2S3Tc 401.51 orthorhombic Pbca 11.0095(4) 12.7034(5) 24.5798(9) 90 90 90 3437.7(2) 8 293(2) 1.552 1.191 1648 0.71073 1.8 × 0.144 × 0.036 1.66-23.29 -10 e h e 12 -14 e k e 14 -27 e l e 25 13310 2467 1.056 R1 ) 0.0426 wR2 ) 0.1036 R1 ) 0.0476 wR2 ) 0.1072

C11H19N2O2S3Tc 405.46 monoclinic P2(1)/c 9.9088(4) 22.2838(8) 7.5060(3) 90 107.6780(10) 90 1579.10(11) 4 293(2) 1.705 1.307 824 0.71073 0.6 × 0.36 × 0.04 1.83-27.98 -13 e h e 13 -25 e k e 28 -9 e l e 8 9262 3579 1.049 R1 ) 0.0411 wR2 )0.1135 R1 ) 0.0436 wR2 )0.1165

thiol) (NS3) was prepared according to standard procedures starting from tris(2-chloroethyl)amine hydrochloride (22). 99Tc as NH4TcO4 was obtained from Amersham as a 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 measured on a SPECORD S10 spectrometer from Carl Zeiss Jena. 1H NMR spectra (400 MHz) were recorded on a Varian spectrometer INOVA-400. Samples were dissolved in deuterated chloroform at a concentration of ca. 2%. Chemical shifts are given as δ in ppm. TLC and HPLC analyses were used to determine the radiochemical purity and stability of the preparations. TLC analyses were performed using silica gel strips (Kieselgel 60) developed with chloroform/acetone (6:1 v/v) as solvents. For HPLC studies, a Perkin-Elmer device consisting of a Turbo LC system with a quarternary pump (Series 200 LC Pump), a programmable absorbance detector model 785A, and a homemade γ-detector (Bohrloch NaI(Tl) crystal) was used. HPLC analyses were carried out with a Hypersil ODS column (250 × 4 mm) using a premixed eluent of 80% methanol and 20% 0.01 M phosphate buffer of pH 7.4 and a flow rate of 1.0 mL/ min. The eluate from the column was monitored by UV absorbance at 254 nm for 99Tc reference complexes or γ 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 graphitemonochromatized Mo KR radiation (λ ) 0.710 73 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 the positions of these, the hydrogen positions were calculated according to ideal geometries.

Empirical absorption corrections were made using ψ 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 2. Atomic positional and thermal parameters, full lists of bond lengths and angles, and Fo/Fc values have been deposited at the Cambridge Crystallographic Data Centre (24). Synthesis of [99Tc(NS3)(PMe2Ph)] - (99Tc1). A 35 mg (0.25 mmol) sample of dimethylphenylphosphine and 20 mg (0.101 mmol) of 2,2′,2′′-nitrilotris(ethanethiol) were dissolved in 3 mL of ethanol. This solution was added to a solution of 20 mg (0.1 mmol) KTcO4 in 2 mL of water/ ethanol (2:1 v/v). The reaction mixture was acidified with three drops of acetic acid and was stirred at ambient temperature for 5 h. During this time, the color changed to deep-violet. The solvents were stripped in vacuo, and the complex was purified by column chromatography (column 10 × 250 mm, stationary phase silica gel 0.040.063 mm, eluent CHCl3/methanol 9:1 v/v). Recrystallization from chloroform/methanol (1:3 v/v) gave violet crystals of 99Tc1 suitable for X-ray structure analysis. Yield: 31 mg (73% relative to KTcO4). Mp ) 221 °C. Anal. (C14H23NPS3Tc) Calcd: C, 39.0; H, 5.3; N, 3.2; S, 22.3. Found: C, 39.1; H, 5.8; N, 3.3; S, 22.6. IR (KBr) (cm-1): 943 and 901 (PhP(CH3)2). UV/vis (CHCl3): λmax(log ) ) 273 nm (4.2), 342 (3.8), 544 (2.9). 1H NMR (400 MHz, CDCl3): 1.91 (d, P(CH3)2Ph, 6H), 2.9 and 3.1 (m, -CH2S, -CH2N, 12H), 7.42 and 7.92 (m, P(CH3)2C6H5, 5H). General Synthesis Procedure for the Complexes [99Tc(NS3)(CNR)] - (99Tc2-99Tc4). A 40 mg (0.093 mmol) sample of 99Tc1 was dissolved in 5 mL of chloroform. At ambient temperature, 0.232 mmol of the appropriate isocyanide, dissolved in 1 mL of chloroform, was added in one portion. The reaction mixture was refluxed for 1 h. TLC showed the presence of the appropriate products 99Tc2-99Tc4 and of unreacted 99Tc1. The isocyanide complexes were isolated by column chromatography (chromatography (column 10 × 250 mm, sta-

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Table 2. Selected Bond Length (Å) and Angles (deg) of Complexes 99Tc1

Tc(1)-N(1) Tc(1)-S(2) Tc(1)-S(1) Tc(1)-S(3) Tc(1)-P(1) N(1)-Tc(1)-S(2) N(1)-Tc(1)-S(1) S(2)-Tc(1)-S(1) N(1)-Tc(1)-S(3) S(2)-Tc(1)-S(3) S(1)-Tc(1)-S(3) N(1)-Tc(1)-P(1) S(2)-Tc(1)-P(1) S(1)-Tc(1)-P(1) S(3)-Tc(1)-P(1)

99Tc2

2.2048(19) 2.2230(6) 2.2258(6) 2.2295(7) 2.3172(6) 85.72(6) 85.97(5) 118.39(3) 85.59(6) 119.46(3) 120.53(3) 176.10(5) 96.78(2) 95.44(2) 90.57(2)

Tc(1)-C(1) Tc(1)-N(2) Tc(1)-S(3) Tc(1)-S(2) Tc(1)-S(1) C(1)-Tc(1)-S(1) N(2)-Tc(1)-S(1) S(3)-Tc(1)-S(1) S(2)-Tc(1)-S(1) C(1)-Tc(1)-N(2) C(1)-Tc(1)-S(3) N(2)-Tc(1)-S(3) C(1)-Tc(1)-S(2) N(2)-Tc(1)-S(2) N(2)-Tc(1)-S(2)

99Tc1, 99Tc2, 99Tc3

and

99Tc4

99Tc3

1.932(10) 2.216(7) 2.227(3) 2.232(2) 2.242(3) 93.8(3) 86.2(2) 120.04(11) 119.18(10) 178.0(3) 95.6(3) 86.1(2) 92.6(3) 85.6(2)

tionary-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 99Tc2, 99Tc3, and 99Tc4 suitable for X-ray structure analysis. [99Tc(NS3)(CNCH2C6H5)] - 99Tc2. Yield: 26 mg (67% relative to 99Tc1). Mp: ) 207 °C. Anal. (C14H19N2S3Tc) Calcd: C, 41.0; H, 4.6; N, 6.8; S, 23.4. Found: C, 40.9; H, 4.3; N, 6.7; S, 22.9. IR (KBr) (cm-1): 2056, 1997 (CN). UV/vis (CHCl3): λmax(log ) ) 278 nm (4.2), 353 (3.7), 520 (2.9). 1H NMR (400 MHz, CDCl3): 2.9 and 3.1 (m, -CH2S, -CH2N, 12H), 5.2 (s, CNCH2, 2H), 7.2 (m, CH2C6H5, 5H). [99Tc(NS3)(CNC6H11)] - 99Tc3. Yield: 23 mg (61% relative to 99Tc1). Mp ) 239 °C. Anal. (C13H23N2S3Tc) Calcd: C, 38.8; H, 5.7; N, 6.9; S, 23.9. Found: C, 39.2; H, 5.8; N, 6.8; S, 23.6. IR (KBr) (cm-1): 2022 (CN). UV/ vis (CHCl3): λmax(log ) ) 277 nm (4.1), 350 (3.7), 527 (2.8). 1H NMR (400 MHz, CDCl3): 1.5 and 1.9 (m, C6H10, 10H), 2.9 and 3.1 (m, -CH2S, -CH2N, 12H), 4.1 (s, CNCH(CH2)2, 1H). [99Tc(NS3)(CNCH2C(O)OC2H5)] - 99Tc4. Yield: 25 mg (65% relative to 99Tc1). Mp ) 194 °C. Anal. (C11H19N2O2S3Tc) Calcd: C, 32.5; H, 4.7; N, 6.9; S, 23.6. Found: C, 32.8; H, 4.9; N, 6.7; S, 23.1. IR (KBr) (cm-1): 2056 (CN), 1746 (CO). UV/vis (CHCl3): λmax(log ) ) 280 nm (4.1), 355 (3.7), 505 (2.8). 1H NMR (400 MHz, CDCl3): 1.32 (t, -OCH2CH3, 3H), 2.8 and 3.2 (m, -CH2S, -CH2N, 12H), 4.6 (q, OCH2-, 2H), 4,8 (s, CNCH2, 2H). General Synthesis Procedure for the No-CarrierAdded Preparation of the Compounds [99mTc(NS3)(CNR)] - (99mTc2-99mTc4). A solution of 20 µL of SnCl2 (1.0-1.5 mg of SnCl2/5.0 mL of 0.1 N HCl) was added to a mixture of 0.5 mL of pertechnetate solution (10-500 MBq generator eluate), 0.3 mL of propylene glycol, 0.2 mL of acetonitrile, 0.3-0.4 mg of 2,2′,2′′-nitrilotris(ethanethiol), dissolved in ethanol, 0.05 mg of the appropriate isocyanide, dissolved in ethanol, and 0.02 mL of 0.1 M NaOH. The vial was closed, and the reaction solution was heated at 50-60 °C for 15 min. The labeling yields for all complexes were between 75% and 80% as established by HPLC analysis. The identity of the species obtained was confirmed by comparison with the HPLC profiles of 99Tc analogues. After HPLC separation the radiochemical purity of the 99mTc(III) “4 + 1” complexes was about 95%. 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 of octanol and shaking for 5 min. After centrifugation aliquots of both phases were counted and the partition coefficient calculated.

99Tc4

Tc(1)-C(1) Tc(1)-N(2) Tc(1)-S(3) Tc(1)-S(1) Tc(1)-S(2) C(1)-Tc(1)-N(2)

1.942(6) 2.184(4) 2.214(2) 2.2153(14) 2.2422(15) 178.7(2)

Tc(1)-C(7) Tc(1)-N(2) Tc1)-S(3) Tc(1)-S(2) Tc(1)-S(1) C(7)-Tc(1)-S(1)

C(1)-Tc(1)-S(3) N(2)-Tc(1)-S(3) C(1)-Tc(1)-S(1) N(2)-Tc(1)-S(1) S(3)-Tc(1)-S(1) C(1)-Tc(1)-S(2) N(2)-Tc(1)-S(2) S(3)-Tc(1)-S(2) S(1)-Tc(1)-S(2)

95.2(2) 86.05(13) 92.8(2) 86.35(12) 117.74(6) 93.9(2) 85.61(12) 120.19(6) 120.62(6)

N(2)-Tc(1)-S(1) S(3)-Tc(1)-S(1) S(2)-Tc(1)-S(1) C(7)-Tc(1)-N(2) C(7)-Tc(1)-S(3) N(2)-Tc(1)-S(3) C(7)-Tc(1)-S(2) N(2)-Tc(1)-S(2) S(3)-Tc(1)-S(2)

1.945(3) 2.199(2) 2.2239(9) 2.2292(9) 2.2357(9) 95.32(10) 85.62(7) 120.22(4) 119.18(4) 178.09(12) 92.18(10) 85.91(7) 95.47(10) 85.50(7) 118.91(4)

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 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% 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), (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 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 5 and 120 min p.i. Selected organs were isolated for weighing and counting. The accumulated radioactivity in the tissue of organs was calculated in terms of percentage of injected dose per organ as well as percent injected dose per gram blood. RESULTS AND DISCUSSION

Technetium-99 Chemistry. The 99Tc complexes Tc2-99Tc4 were synthesized by a two-step procedure starting from pertechnetate(VII) via the phosphinecontaining precursor 99Tc1 according to Scheme 1. To a mixture of pertechnetate(VII) and 2,2′,2′′-nitrilotris(ethanethiol) (NS3) was added an excess of dimethylphenylphosphine. The phosphine acts as a reducing agent as well as a coligand to yield 99Tc1. The complex can be isolated as an air-stable deep-violet powder. This Tc(III) phosphine derivative undergoes in chloroform solutions facile substitution to the corresponding neutral “4 + 1”coordinated Tc(III) complexes 99Tc2-99Tc4 of the general formulation [Tc(NS3)(CN-R] (Scheme 1). Elemental analyses, as reported in the Experimental Section, are in agreement with the proposed formulation. IR spectra of Tc(III) complexes 99Tc2-99Tc4 exhibit 99



Scheme 1

strong absorptions characteristic of the Tc-CN- stretching vibrations. No additional bands indicating the presence of the TcdO core in the region 900-1000 cm-1 are observed. The UV/vis spectra of 99Tc2, 99Tc3, and 99Tc4 (recorded in chloroform) are characterized by intense bands at about 280 and 350 nm and less intense absorptions in the visible region (505-527 nm). The Tc(III) complexes show sharp proton signals characteristic of diamagnetic compounds, in agreement with low-spin d4 tbp configurations. NMR profiles confirm the solution structures of complexes 99Tc1-99Tc4 to be identical to solid-state structure found by X-ray analysis. Five-coordinate Tc(III) complexes 99Tc1-99Tc4, 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, and selected bond lengths and angles are cumulated in Table 2. As illustrated in Figures 1-4, the complexes adopt a nearly ideal trigonal-bipyramidal geometry with the trigonal plane formed by the three thiolate sulfurs of the tripodal ligand. The central nitrogen atom of the chelate ligand and the phosphine phosphorus in 99Tc1 or the monodendate isocyanide ligand in 99Tc2-99Tc4 occupy the apical positions. The Tc-Sthiolato distances are restricted in a narrow range (2.21-2.24 Å) and compare well with those previously reported for trigonal bipyramidal [Tc(SR)3(L)2]-type complexes (SR ) sterically hindered arenethiolates, L ) small π-accepting molecules) (25, 26). The Tc-C distances are found in a narrow range between 1.93 und 1.94 Å. The N2-Tc-C1 axis shows only slight deviation from linearity. Also the other angles of the inner coordination sphere are consistent with the tbp arrangement.

Figure 1. Molecular structure of complex

99Tc1.


99Tc2.


99Tc3.


99Tc4.

The compounds constitute the second example of an Tc(III) complex with an “umbrella” ligand, a concept introduced by Davison and co-workers (27, 28). The “umbrella” in Davison’s complexes of the general formula [Tc(PS3)(CN-R)] consists of a central phosphorus atom and three aromatic thiolate functions (PS3), while in 99mTc2-99mTc4 three aliphatic thiolates and a central nitrogen form the “umbrella” ligand (NS3). Technetium-99m Chemistry and Biodistribution Studies. No-Carrier-Added Preparation of the 99mTc Complexes. The no-carrier-added preparation of the complexes 99mTc2-99mTc4 was performed by a one-step procedure starting from 99m[TcO4]- with stannous chloride as reducing agent (Scheme 1). The yields were between 75% and 80% using unoptimized preparation conditions. In the appropriate reaction mixtures, a byproduct occurs that is not eluted from the Hypersil column. Therefore, TLC has to be used for determining the yields of the preparation. After HPLC separation, the

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Table 3. HPLC and TLC Dataa of Complex Couples 99Tc2/99mTc2, 99Tc3/99mTc3, and 99Tc4/99mTc4 (Partition Coefficients (log P) of 99mTc2-99mTc4 in Comparison to the Data of Corresponding “3 + 1” Mixed-Ligand Oxotechnetium(V) Complexes [TcO(SCH2CH2N(CH3)CH2CH2S)(SR)] Bearing the Same Substituent R (A-C)) complex 99Tc2 99mTc2

tR (min) 5.8 5.9

A 99Tc3 99mTc3

7.1 7.3

B 99Tc4 99mTc4

C

7.7 7.7

log P (octanol/saline) 1.5 1.0 1.7 0.6 1.6 0.5

Rf 0.4 0.4 0.3 0.3 0.1 0.1

a

Hypersil ODS: MeOH/0.01 M PBS pH 7.4, linear gradient 70100% A in 5 min, TLC data: TLC aluminum sheets RP-18 (Merck); eluent, methanol/water (80/20).

radiochemical purity of the 99mTc(III) “4 + 1” complexes was determined by TLC and found to be about 95%. 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 complexes 99mTc2 and 99Tc2, practically identical retention times were observed, while the recovery through the column was quantitative. Similarly, co-injections of the complex couples 99mTc3/99Tc3 and 99mTc4/99Tc4 led to identical tR values for the respective compounds, revealing their structural analogy. tR values of HPLC, Rf values of TLC, and partition coefficients (P) of the 99mTc complexes in the system octanol/saline are given in Table 3. Examination of the octanol/water partition coefficients suggests high lipophilicity for the resulting Tc species. A comparison with the values of so-called “3 + 1” mixedligand oxotechnetium(V) complexes A-C of the general formula [TcO(SCH2CH2N(Me)CH2CH2S)(SR)] (23) bearing the same substituent R (complex (A) benzyl, (B) cyclohexyl, (C) carbethoxymethylene) indicates considerably higher lipophilicity of the studied “4 + 1” complexes. These observations are consistent with the expectation that absence of an oxo group and better shielding of the metal will enhance the lipophilicity (Table 3). Glutathione Challenge Experiments. Glutathione (GSH), the most abundant thiol compound in tissues, is present in almost all animal cells in relatively high concentrations (0.5-12 mM) (29, 30). Therefore, it has to be considered as a potential agent for transchelating reactions in vivo. In fact, Neirinckx et al. (31) pointed out that the intracellular reaction of the brain perfusion agent 99mTc(V)-HMPAO with GSH is responsible for the trapping of the radiopharmaceutical in the brain. GSH is also responsible for the in vivo reactivity of “3 + 1” mixed-ligand 99mTc(V) complexes that consist of a monodentate thiol ligand and a tridentate dithiol (9-11). Here, a ligand exchange in terms of the replacement of the monodentate ligand by GSH occurs in vivo and in vitro as recently shown (12, 13, 32). In view of these experiences, it would be useful to explore also the behavior of 99mTc(III) “4 + 1” complexes against GSH ligandexchange reactions. Saline-containing preparations of the 99mTc compounds were diluted with a 10 mM GSH solution immediately after HPLC purification. In all cases, no additional peaks of Tc-GSH mixed ligand species were observed in HPLC studies. These results clearly indicate that glutathione has no negative effect on the stability of 99mTc complexes in solutions. Obviously, no transchelation reaction by

Figure 5. HPLC pattern of the complex 99mTc4; 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)]: (A) 120 min incubation in rat erythrocyte hemolysate; (B) 120 min incubation in rat plasma.

GSH occurs in vitro. This behavior is considered to be an important advantage of the 99mTc(III) “4 + 1” complexes over mixed-ligand 99mTc oxocomplexes in the oxidation state +5. Stability Studies in Plasma and in Rat Blood. For stability studies, the 99mTc complexes were incubated in rat blood and rat plasma for 120 min. After incubation, the blood was centrifuged and the supernatant plasma was separated from the erythrocytes. The plasma as well as the erythrocyte hemolysate were analyzed using a Supelguard column as described above. For the complexes 99mTc2-99mTc4 no additional peaks were observed, indicating some kind of decomposition, modification, or alteration of the complexes. There is also no tendency of these complexes to bind on plasma components. To confirm these results, the plasma samples as well as the hemolysates were extracted with ethyl acetate. In this way, about 95% of the activity could be extracted. Analyzing the extracts by HPLC using a Hypersil column and by TLC as described in the Experimental Section only the parent compounds were detected. These results indicate that the 99mTc(III) “4 + 1” complexes behave absolutely stable in plasma and blood of rat for at least 120 min as illustrated for 99mTc4 in Figure 5. Biodistribution Studies in Rats. Data of the biodistribution studies of 99mTc complexes 99mTc2-99mTc4 are shown in Table 4. All three complexes show low radioactivity in blood at 5 min p.i., which thereafter decreases within 120 min to a similar extent. The neutral, lipophilic



Table 4. Biodistribution Pattern of Complexes means ( SD; n ) 5)

99mTc2-99mTc4

in Wistar Rats (% ID/Organ Except for Blood as % ID/g;

complex

min p.i.

blood

brain

heart

lungs

kidneys

liver

99mTc2

5 120 5 120 5 120

0.34 ( 0.09 0.11 ( 0.04 0.23 ( 0.11 0.13 ( 0.07 0.13 ( 0.02 0.07 ( 0.01

0.79 ( 0.11

Mixed-Ligand Technetium(III) Complexes with Tetradendate

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