Modified â4 + 1â Mixed Ligand Technetium-Labeled Fatty Acids for

Bioconjugate Chem. 2008, 19, 97–108

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Modified “4 + 1” Mixed Ligand Technetium-Labeled Fatty Acids for Myocardial Imaging: Evaluation of Myocardial Uptake and Biodistribution Peter Mirtschink,*,† Sebastian N. Stehr,§ Hans J. Pietzsch,‡ Ralf Bergmann,‡ Jens Pietzsch,‡ Gerd Wunderlich,| Anke C. Heintz,§ Joachim Kropp,⊥ Hartmut Spies,+ Werner Kraus,# Andreas Deussen,† and Martin Walther‡ Institute of Physiology, Department of Anesthesiology, and Department of Nuclear Medicine, Technical University Dresden, D-01307 Dresden, Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, D-01314 Dresden, Department of Nuclear Medicine Carl Thiem-Hospital Cottbus, D-03048 Cottbus, and Federal Institute for Materials Research and Testing (BAM), D-12205 Berlin, Germany. Received May 7, 2007; Revised Manuscript Received September 3, 2007

Our group previously synthesized 99mTc-labeled fatty acids suitable for myocardial metabolism and flow imaging. In this set of experiments, 29 new analogues were synthesized according to the “4 + 1” mixed ligand approach with some specific differences. Conventional “4 + 1” 99mTc-fatty acids are built in the sequence: Tc-chelate, alkyl chain, and carboxylic group. We developed compounds following a new design with the sequence: carboxylic group, alkyl chain, Tc-chelate, and lipophilic tail. Therefore, the 99mTc-chelate was transferred to a more central position of the compound, aiming toward an improved myocardial profile and an accelerated liver clearance. In this context, several functional groups incorporated in the lipophilic tail section were tested to evaluate their influence on the compound’s character. In addition to biodistribution studies in ViVo, the myocardial first-pass extraction of the compounds was tested in an isolated Langendorff rat heart model. A satisfactory myocardial uptake of up to 20% of the injected dose (% ID) in the perfused heart and a fast liver clearance in ViVo with only 0.29% ID/g at 60 min postinjection demonstrate that the induced molecular modifications affect the kinetics of 99m Tc-radiolabeled fatty acid compounds favorably. From the data set, rules for estimating the biodistribution of fatty acids tracers are deduced.

INTRODUCTION The development of 99mTc-labeled fatty acids (FA) suitable for evaluation of myocardial function and metabolism has been studied by various research groups since 1975 (1). The greatest challenge has been proven to be the design of ligands, which offer a stable binding between the FA and the metal core. Simultaneously, these ligands should not interfere with the biological properties of the FA, allowing an unimpeded recognition by membranous and intracellular proteins. Of the broad variety of neutral and small-sized mixed ligand chelate types, square-pyramidal Tc(V) oxocomplexes of tetradentate ligands are most commonly used (2–9). Because of disappointing results in biodistribution studies, a strong interference with the metal oxo-unit was assumed, influencing the molecular properties and the in ViVo behavior of FA. Besides these types, lipophilic Tc(I) species were evaluated (10, 11). After gaining experiences in FA labeling with oxotechnetium(V) complexes (12, 13), we focused on the development of “4 + 1” mixed ligand Tc(III) FA derivatives (13, 14). The presently available evidence suggests that in the “4 + 1” donor atom arrangement the radiometal is well-shielded by both a tripodal tetradentate ligand and a monodentate ligand bearing the FA moiety (15). * Corresponding author: Peter Mirtschink, Institute of Physiology, Medical Faculty Carl Gustav Carus Technical University of Dresden, Fetscherstr. 74 01307, Dresden, Germany. Tel.: +49 351 458 6014. Fax: +49 351 458 6301. E-mail: [email protected]. † Institute of Physiology, Technical University Dresden. § Department of Anesthesiology, Technical University Dresden. ‡ Institute of Radiopharmacy. | Department of Nuclear Medicine, Technical University Dresden. ⊥ Department of Nuclear Medicine Carl Thiem-Hospital Cottbus. # BAM. + Deceased on March 25, 2007.

In experiments using the isolated guinea pig heart, the evaluated compounds clearly outmatched the extraction results of the “3 + 1” oxotechnetium(V) FA analogues. Furthermore, no in Vitro transchelation reaction with glutathione was observed, indicating a high stability toward those challenging agents (15). Although promising uptake results were achieved with the isolated heart model, no “4 + 1” FA compound synthesized so far had similarly advantageous biodistribution parameters to those of established iodinated FA tracers (e.g., iodine-labeled 15-(p-iodophenyl)pentadecanoic acid, 123I-IPPA, and 15-(piodophenyl)-3-(R,S)-methylpentadecanoic acid, 123I-BMIPP). Therefore, the main problems, namely, the high liver uptake, as observed for many other 99mTc-labeled FA compounds, and a decelerated liver clearance, remained. In an attempt to synthesize 99mTc-labeled FA based on the promising “4 + 1” mixed ligand approach with an improved myocardial profile and a faster liver washout, some innovative structural modifications were introduced. While the conventional “4 + 1” 99mTc-FA are built in the sequence Tc-chelate, alkyl chain, and carboxylic group, modified compounds were designed according to the sequence carboxylic group, alkyl chain, Tcchelate, and lipophilic tail. Therefore, the 99mTc-chelate was transferred to a more central position, to avoid nonrecognition by membranous and intracellular enzymes because of a terminally positioned artificial 99mTc-chelate. In the following, we describe the chemistry of modified “4 + 1” 99mTc-FA as well as some improvements and adaptation of the recently published labeling procedure (14). Moreover, we correlate changes in molecular structure of the synthesized 99m Tc compounds with biological behavior, namely, uptake and biodistribution. For better clarity, Tc complexes and their corresponding Re compounds were organized into groups (denoted by I-VII and ReI-ReVII or rather ReMeI-ReMeVII

10.1021/bc700164c CCC: $40.75  2008 American Chemical Society Published on Web 12/04/2007

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Figure 1. The compound groups I–VII of the recent “4 + 1”-99mTc/ Re FA complexes with different metabolic stability depending on the chemical properties of the monodentate ligand. All compounds are shown in Table 1. Unfunctionalized isocyanides in Ia–Ie. Unfunctionalized phosphines in IIa–IId. Phosphines or phospinites in IIIa–IIIb. Isocyano alkylamides in IVa–VIe. Alkyl isocyano alkanoates in Va–Vh. Carboxyl group containing phosphines or isocyanides in VIa–VIc. Unsubstituted tetradentate ligand fatty acid containing phosphine in VII.

Figure 2. Synthesis of the FA substituted tristhiolate ligand.

for the corresponding Re(methyl) derivatives) according to their physicochemical properties determined by the monodentate ligands (Figure 1).

EXPERIMENTAL PROCEDURES Materials and Instrumental Techniques. EDTA was purchased from Riedel-deHaen. PhPMe2 (M0), PPh3 (M1), Ph2Pp-C6H4COOH (M2), P(PropCN)3 (M3), P(o-Tol)3 (M4), Ph2POEt (M5), and trifluoroacetic acid (TFA) were purchased from Aldrich. 2-Isocyano-2-methylpropane (or TBI t-butyl isonitril) (M7) were purchased from Alfa products and a commercial Cardiolite kit with 1-isocyano-2-methoxy-2-methylpropane (or MIBI 2-methoxyisobutyl isonitril) (M8) from Bristol-Myers-Squibb. All commercial reagents were used without further purification. 2,3,5,6-Tetrafluorophenyl 4-isocyanobutanoate and 2,3,5,6-tetrafluorophenyl 4-(isocyanomethyl) benzoate, serving as precursors for the monodentate isocyano ligand, and 3-(benzylthio)-2-{bis-[2-(benzylthio)ethyl]amino} propanoic acid (BzNS3COOH) were prepared as described (16). The alkyl (isocyano) alkanoates ethyl 8-isocyanooctanoate (M12), ethyl 4-isocyanobutyrate (M13), ethyl 2-isocyanoacetate (M14), methyl 15-isocyanopentadecanoate (M15-Me), methyl 5-[(7-isocyanoheptyl)thio]pentanoate (M16-Me) and methyl 12-

Mirtschink et al.

isocyanododecanoate (M19-Me) were prepared according the procedure that was used before (14) (see Figure 1 in Supporting Information for detail). Proton nuclear resonance (1H NMR) spectra were recorded on a 400 MHz Varian Inova 400 spectrometer (1H 400 MHz, 13 C 100 MHz, 31P 162 MHz). The 1H and 13C chemical shifts are reported in parts per million (ppm) relative to residual solvent signals or TMS as reference. 31P chemical shifts are reported relative to H3PO4 as an external reference. Mass spectrometric measurements were performed with a Micromass Tandem Quadropole Mass Spectrometer (Quadro LC) operating in the MS mode. Mass spectral data were recorded in the positive or negative ESI mode. About 10-4 M of the sample dissolved in 1.0 mL of methanol or acetonitrile was injected at a flow rate of 5 mL/min. Elemental analyses were performed on a LECO Elemental Analyzer CHNS-932. UV/vis spectra were recorded on a Varian Cary 50 Bio spectrometer. HPLC analysis and purification of 99mTc-complexes were performed using a PerkinElmer instrument consisting of a Turbo LC System with a quaternary pump (series 200 LC pump) and a programmable absorbance detector model 785A. Infrared spectra were recorded on a FTIR-Spectrophotometer Spectrum 2000 (Perkin-Elmer). Determination of melting points was carried out with the help ¨ CHI Melting Point B-540 machine. Analytical thin layer of a BU chromatography was performed on POLYGRAM SIL G/UV254 foils (Machery-Nagel). Nonradioactive rhenium complexes were monitored by their UV absorption at 254 nm, and 99mTccomplexes by gamma-ray detection (Bohrloch, NaI(Tl) crystal). Synthesis of Tetradentate NS3-Ligands (T3-T5).General Coupling to FA. DCC (433 mg, 2.1 mM), dissolved in 5 mL dichloromethane, was added dropwise at 0 °C to a solution of 3-(benzylthio)-2-{bis-[2-(benzylthio)-ethyl]-amino}-propanoic acid [(BzNS3COOH) 1074 mg, 2.1 mM] in 10 mL dichloromethane. After stirring for 15 min, 1-hydroxybenzotriazole (284 mg, 2.1 mM) was added. Subsequently, the alkyl-ω-aminoalkanoate (2.5 mM) dissolved in 10 mL dichloromethane was added, after the deliverance of the amine in the organic phase via extraction with a saturated solution of potassium carbonate from the corresponding hydrochloride. The reaction mixture was stirred first for 2 h at 0 °C, then 12 h at room temperature. After filtering off the precipitated byproduct, N,N′-dicyclohexylurea, the solvent was removed in vacuo. The purification of the yellow, oily crude product was performed chromatographically (n-hexane/ethyl acetate ) 1/1, Rf ) 0.6–0.85 depending on chain length) and yielded a slightly yellow, viscous oil (selected NMR data are shown in Supporting Information). To the alkyl (BzNS3CONH-) alkanoate (0.25 mM) dissolved in 10 mL dioxane was added 4 mL 1 N aqueous sodium hydroxide for the saponification of the ester function. After stirring overnight, the reaction mixture was acidified with 1 N aqueous hydrochloric acid. This step was necessary, because the reducing conditions in the following deprotection step led to alkanoles in the case of esters. After acidification with TFA to pH 3–4 and evaporation of the solvent, a 3-fold extraction with chloroform/water followed. The united organic phases were dried with MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (n-hexane/ethyl acetate ) 1/1, Rf ) 0.55–0.65) (selected NMR data are shown in Supporting Information). Deprotection of the Tris-Thiolate Ligand. The FA containing BzNS3CONHR-ligand (0.49 mM) was dissolved in 3 mL waterfree THF and added dropwise to liquid ammonia at -50 °C. Under gentle stirring, pieces of sodium were added. The termination of the reaction was seen in the excess of sodium, giving the reaction mixture a deep blue color. Subsequently, the evaporation of the solvent followed in a nitrogen gas stream. The remaining solid was carefully dissolved in 10 mL water,

Synthesis and Evaluation of Technetium-Labeled Fatty Acids

acidified with 1 N hydrochloric acid (to pH 3–4), and extracted 5 times, each with 7 mL ethyl acetate. The product was obtained by concentration of the organic phases in vacuo and precipitation of the product with hexane. Because of the product instability, no more purification steps were performed. The syntheses of the unsubstituted tetradentate ligands (TX), the oxalic acid salt of tris(2-mercaptoethyl)amine (T1), and 1-carboxy-3-mercapto-N,N-bis(2-mercaptoethyl)-1-oxopropan2-aminium chloride (T2) have been described recently (16, 17). 1-[(7-carboxyheptyl)amino]-3-mercapto-N,N-bis(2-mercaptoethyl)-1-oxopropan-2-aminium chloride (T3). 1H NMR (CD3CN): 1.31 (m, 6H, CH2), 1.46–1.57 (m, 4H, CH2), 2.28 (t, 2H, CH2COO, 3J ) 8.0 Hz), 2.63 (t, 2H, CONHCH2, 3J ) 8.0 Hz), 3.14–3.21 (m, 10H, 2 × NCH2CH2SH, NCH2), 3.35–3.37 (m, 1H, CHCONH). 13C NMR (CD3CN): 22.1, 23.0, 23.5, 24.8, 26.7, 28.9, 29.4 (2C), 33.5, 39.0, 54.2, 54.8, 72.3, 169.2, 175.1. IR (KBr): 3410 (COOH, CONH), 2928, 2853 (CH2), 1715 (COOH), 1648 (CONH). ESI-MS (MeOH): m/z (%) ) 383 (90) for [C15H31N2O3S3]+. 1-[(11-carboxyundecyl)amino]-3-mercapto-N,N-bis(2-mercaptoethyl)-1-oxopropan-2-aminium chloride (T4). 1H NMR (CDCl3): 1.21–1.27 (m, 14H, CH2), 1.49–1.54 (m, 4H, CH2), 2.28 (t, 2H, CH2COO, 3J ) 7.8 Hz), 2.60 (t, 2H, CONHCH2, 3 J ) 7.9 Hz), 3.00–3.28 (m, 10H, 2 × NCH2CH2SH, NCH2). 3.33–3.36 (m, 1H, CHCON). 13C NMR (CDCl3): 22.3, 23.9, 25.9, 27.2, 29.2, 29.3, 29.4 (2C), 29.5 (2C), 29.6, 29.7 (2C), 32.9, 39.9, 54.5, 67.1, 72.9, 169.5, 176.8. IR (KBr): 3426 (COOH, CONH), 2926, 2853 (CH2), 1710 (COOH), 1645 (CONH). ESI-MS (MeOH): m/z (%) ) 439 (35) for [C19H39N2O3S3]+. 1-[(14-carboxytetradecyl)amino]-3-mercapto-N,N-bis(2-mercaptoethyl)-1-oxopropan-2-aminium chloride (T5). 1H NMR (CDCl3): 1.20–1.27 (m, 20H, CH2), 1.50 (m, 4H, CH2), 2.28 (t, 2H, CH2COO, 3J ) 8.0 Hz), 2.62 (t, 2H, CONHCH2, 3J ) 7.9 Hz), 3.00–3.30 (m, 10H, 2 × NCH2CH2SH, NCH2). 3.36 (m, 1H, CHCON). 13C NMR (CDCl3): 22.0, 22.9, 25.5, 26.0, 26.9, 28.9, 29.2, 29.4 (2C), 29.5 (4C), 29.6, 29.7 (2C), 33.5, 39.9, 54.5, 54.6, 72.7, 169.5, 177.2. ESI-MS (MeOH): m/z (%) ) 481 (90) for [C22H45N2O3S3]+. Syntheses of Monodentate Ligands MY (M6, M9–M11, and M17–M18).Phosphorus(III) Ligands. The phosphorus(III) ligands M0–M5 were commercial reagents and were used without further purification. 12-{[(Diphenylphosphino)acetyl]amino}dodecanoic acid (M6). The coupling of 12-aminododecanoic acid with diphenyl phosphine acetate was carried out with DCC as described above for BzNS3CONHR, the precursors for T3–T5. DCC (43 mg, 0.21 mM), dissolved in 1 mL dichloromethane, was added dropwise at 0 °C to a solution of diphenyl phosphine acetate (49 mg, 0.2 mM) in 2 mL dichloromethane. After stirring for 15 min, 1-hydroxybenzotriazole (27 mg, 0.2 mM) was added first, followed by the 12-amino-dodecanoic acid (43 mg, 0.2 mM) suspended in 5 mL dichloromethane. The reaction mixture was stirred for 2 h at 0 °C, then for 12 h at room temperature. After filtering off the precipitated byproduct N,N′-dicyclohexylurea and unreacted amino acid, the solvent was removed in vacuo. Because of the product instability toward oxygen, no more purification steps were performed. Yield: 67 mg (76%); colorless waxy solid. 1H NMR (CDCl3): broad signals, because of an equilibrium of tautomeric species, 1.12–1.37 (m, 14H), 1.56 (m, 2H), 1.60 (m, 2H), 2.18 (m, 2H), 3.10 (m, 2H), 3.53 (m, 2H, PC H2CONH), 6.6 (s, 1H, NH), 7.30 (m, 2H, phenyl), 7.38 (m, 4H, phenyl), 7.80 (m, 4H, phenyl). 13C NMR (CDCl3): 25.2, 25.9, 26.3, 26.9, 29.5 (3C), 34.2, 39.9, 49.1, 63.5, 129.0 (4C), 130.9 (2C), 132.6 (2C), 169.3 (CONH), 179.8 (COO). 31 P NMR (CDCl3): -16.3. ESI-MS (MeOH): m/z (%) ) 465 (100) for [C26H36NNaO3P]+.

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Isocyano Ligands M9–M11 and M17–M18.Isocyano Alkylamide Ligands M9–M11.General Procedures. 0.10 mM of the amine, dissolved in 1 mL THF, was added dropwise to 2,3,5,6-tetrafluorophenyl 4-isocyanobutanoate (0.10 mM) and 2,3,5,6-tetrafluorophenyl 4-(isocyanomethyl)-benzoate (0.10 mM) dissolved in 1 mL THF. The reaction mixture was stirred for 2.5 h at room temperature. Then, separation of the solvent in vacuo and purification by column chromatography followed. 4-Isocyano-N-octylbutanamide (M9). After removal of the solvent, the crude product was purified by column chromatography (silica gel 60, tert-butyl methyl ether/n-hexane ) 2/1, Rf ) 0.10). Yield from 12.9 mg (0.10 mM) 1-aminooctane: 14.5 mg (65%) colorless oil. 1H NMR (CDCl3): 0.86 (t, 3H, CH3, 3J ) 7.0 Hz), 1.25–1.28 (m, 10H, CH2), 1.48 (m, 2H, CH2), 1.95–2.03 (m, 2H, CH2), 2.33 (t, 2H, CH2CONH, 3J ) 7.0 Hz), 3.22 (m, 2H, NHCH2), 3.47 (tt, 2H, CNCH2, 3JHH ) 6.2 Hz, 2 JHN ) 1.7 Hz), 5.56 (s, 1H, CONH). 13C NMR (CDCl3): 14.3, 22.8, 24.9, 27.1, 29.4, 29.5, 29.8, 32.0, 32.5, 39.8 (NHCH2), 41.2 (t, CNCH2, 2JCN ) 6.5 Hz), 156.5 (t, CN, 1JCN ) 5.8 Hz), 171.0. IR (KBr): 3313, 2957, 2927, 2856 (CH2, CH3), 2148 (CN), 1642, 1554. ESI-MS (MeOH): m/z (%) ) 225 (100) for [C13H25N2O]+. N-butyl-4-isocyano-N-methylbutanamide (M10). Column chromatography (silica gel 60; tert-butyl methyl ether/n-hexane ) 4/1, Rf ) 0.40. Yield: (70%) colorless oil. 1H NMR (CDCl3): particular doubling of signals because of the mixture of cis/ trans isomers, 0.90 (t, 3H, CH3, 3J ) 7.2 Hz), 0.93 (t, 3H, CH3, 3 J ) 7.0 Hz), 1.21–1.36 (m, 4H, CH2), 1.42–1.57 (m, 4H, 2 × CH2), 1.92–2.03 (m, 4H, CH2), 2.44 (t, 2H, CH2CONH, 3J ) 6.8 Hz), 2.47 (t, 2H, CH2CONH, 3J ) 6.8 Hz), 2.89 (s, 3H, NCH3), 2.96 (s, 3H, NCH3), 3.25 (t, 2H, NCH2, 3J ) 7.6 Hz), 3.33 (t, 2H, NCH2, 3J ) 7.6 Hz), 3.50 (tt, 4H, CNCH2, 3JHH ) 6.2 Hz, 2JNH ) 1.9 Hz). 13C NMR (CDCl3): 14.0, 14.1, 20.2, 20.3, 24.5, 24.7, 28.7, 29.4, 29.6, 30.7, 33.6 (N CH3), 35.3 (N CH3), 41.4 (t, CNCH2, 2JCN ) 6.2 Hz), 41.5 (t, CNCH2, 2JCN ) 6.1 Hz), 47.8, 49.8, 156.2 (broad, CN), 170.8. ESI-MS (MeOH): m/z (%) ) 183 (95) for [C10H19N2O]+. 4-(Isocyanomethyl)-N-octylbenzamide (M11). Column chromatography (silica gel 60, tert-butyl methyl ether/n-hexane ) 2/1, Rf ) 0.34). Yield: (91%) colorless solid. Mp 82 °C. 1H NMR (CDCl3): 0.86 (t, 3H, CH3, 3J ) 6.6 Hz), 1.24–1.37 (m, 10H, CH2), 1.60 (m, 2H, CH2), 3.43 (t, 2H, NHCH2, 3J ) 7.1 Hz), 4.68 (s, 2H, CNCH2Ph), 6.23 (s, 1H, CONH), 7.38 (d, 2H, phenyl, 3J ) 8.4 Hz), 7.78 (d, 2H, phenyl, 3J ) 6.4 Hz). 13 C NMR (CDCl3): 14.3, 22.9, 29.4, 29.5, 29.9, 31.8, 32.0, 40.4, 45.5 (t, CNCH2Ph, 2JCN ) 6.9 Hz), 126.9 (2C), 127.8 (2C), 135.3, 135.6, 156.8 (t, CN, 1JCN ) 5.6 Hz), 166.9. IR (KBr): 3348, 3063 (CH-phenyl), 2954, 2921, 2851 (CH2, CH3), 2158 (CN), 1632. ESI-MS (MeOH): m/z (%) ) 273 (100) for [C17H25N2O]+. Alkyl isocyanoalkanoate. Preparation according the other described alkyl-(isocyano)alkanoates (14). Octyl isocyanoacetate (M17). Yield: (86%) viscous colorless oil. 1H NMR (CDCl3): 0.86 (t, 3H, CH3, 3J ) 6.0 Hz), 1.25–1.35 (m, 10H, CH2), 1.61–1.69 (m, 2H, CH2), 4.18–4.20 (m, 4H, CNCH2+ OCH2). 13C NMR (CDCl3): 14.3, 22.8, 25.9, 26.0, 28.6, 29.3, 31.9, 43.7, 67.1, 157.4 (broad), 164.2. ESI-MS (MeOH): m/z (%) ) 198 (100) for [C11H20NO2]+. Dodecyl Isocyanoacetate (M18). Yield: (89%) viscous colorless oil. 1H NMR (CDCl3): 0.84 (t, 3H, CH3, 3J ) 6.2 Hz), 1.20–1.36 (m, 18H, CH2), 1.60–1.68 (m, 2H, CH2), 4.16–4.19 (m, 4H, CNCH2+ OCH2).13C NMR (CDCl3): 14.2, 22.6, 25.8, 26.1, 28.5, 29.1 (2C), 29.3 (2C), 29.6, 31.8, 43.5, 67.0, 156.8 (broad), 166.4. ESI-MS (MeOH): m/z (%) ) 254 (100) for [C15H28NO2]+.


Preparation of Selected 185/187Rhenium and 99gTechnetium Complexes as Reference Compounds. “4 + 1” FA-NS3 Redimethylphenylphosphine derivatives (TX-Re-M0) were used in ligand exchange reactions as ideal FA precursor complexes for the preparation of the corresponding isocyano-substituted compounds. [Methyl 12-({N,N-bis[2-(mercapto-KS)ethyl]cysteinyl-K2 2 3 N ,S }amino)dodecanoate](dimethylphenylphosphine)rhenium (III) (MeT4-Re-M0). The tripodal ligand 1-[(11-carboxyundecyl)amino]-3-mercapto-N,N-bis(2-mercaptoethyl)-1-oxopropan2-aminium chloride (T4) (100 mg, 0.21 mM) and the monodentate ligand dimethyl phenyl phosphine (M0) (58 mg, 0.42 mM) were refluxed for 3 h in methanol with the hexathiourea Re(III) complex {Re[SC(NH2)2]6}Cl3 × 1H2O (161.5 mg, 0.21 mM) as an appropriate Re(III) precursor. The hydrochloric acid formed during the reaction in the methanol leads to esterification of the carboxyl group (MeT4). (This esterification does not happen with the no carrier added (nca) preparation of the appropriate 99mTc compounds in predominantly aqueous medium.) After removal of the solvent, the crude product was purified by column chromatography (silica gel 60, chloroform/ ethyl acetate, 4/1, Rf ) 0.50). Yield: 75 mg (46%) green solid. 1 H NMR (CDCl3): 1.22–1.31 (m, 14H, CH2), 1.50 (m, 2H, CH2), 1.59 (m, 2H, CH2), 2.07 (d, 6H, PCH3, 3J ) 3.2 Hz), 2.28 (t, 2H, CH2COO, 3J ) 7.4 Hz), 2.33–2.41 (m, 1H, NCH2), 2.68–2.83 (m, 6H), 3.03–3.30 (m, 7H), 3.35–3.38 (m, 1H, NCH2), 3.63 (s, 3H, OCH3), 3.65–3.73 (m, 1H, CHCONH), 5.98 (t, 1H, CONH, 3J ) 5.6 Hz), 7.25–7.29 (m, 1H, phenyl), 7.37–7.41 (m, 2H, phenyl), 7.70–7.75 (m, 2H, phenyl). 13C NMR (CDCl3): 25.1, 27.1, 28.9, 29.1 (2C), 29.3, 29.4 (2C), 29.5, 29.6 (2C), 34.3 (CH2COO), 40.1 (CONHCH2), 47.3, 47.4, 50.1, 51.4, 51.7 (O CH3), 73.0 (CHCONH), 128.2 (2C), 128.9, 130.5 (2C), 130.6, 169.1 (CONH), 174.7 (CH2COOR). 31P NMR (CDCl3): –13.4. IR (KBr): 3070, 3052 (phenyl), 2925, 2853 (CH2, CH3), 1737 (COOR), 1676 (CO). UV/vis (CHCl3): λmax (nm): 335.6, 465.4. [12-({N,N-Bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino) dodecanoato][2-(isocyano-KC)-2-methylpropane]rhenium(III) (ReIb). The Re-dimethylphenylphosphino precursor complex (MeT4-Re-M0) (10 mg, 0.0129 mM) was stirred with 2-isocyano-2-methylpropane (M7) (2 mg, 0.026 mM) for 2 h at room temperature in 2 mL chloroform. After an additional step for ester saponification and removal of the solvent, the crude product was purified by column chromatography (silica gel 60; chloroform/ethyl acetate ) 4/1, Rf ) 0.65). Yield: 8.6 mg (95%) green solid. 1H NMR (CDCl3): 1.21–1.36 (m, 14H, CH2), 1.44 (s, 9H, CCH3), 1.48–1.80 (m, 4H, CH2), 3.44 (m, 1H, CH2), 3.64 (m, 1H, CH2), 3.78 (m, 1H), 3.94 (m, 1H, CHCON), 6.82 (s, broad, 1H, CONH). 13C NMR (CDCl3): 24.8, 25.5, 25.7, 26.6, 26.9, 28.4, 29.3, 29.4 (2C), 29.7, 31.0, 31.5, 32.9, 36.2, 40.1, 47.1, 49.7, 56.2, 71.2, (NCHCO), 164.9 (CNC(CH)3), 169.5 (CONH), 179.2 (COOH). ESI-MS (MeOH): m/z (%) ) 705 (100) for [C24H45N3O3ReS3]+. [8-({N,N-Bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino)octanoic acid](triphenylphosphine)rhenium(III) (ReIIa). Prepared according the procedure for MeT4-Re-M0 under the use of T3 (19 mg, 0.05 mM), the hexathiourea Re(III) complex (38 mg, 0.05 mM) and M1 (26 mg, 0.1 mM) and an additional step for ester saponification. Column chromatography (silica gel 60, chloroform/ethyl acetate ) 2/1, Rf ) 0.3). Yield: 28 mg (69%) green solid. 1H NMR (CD3CN): 1.27–1.37 (m, 6H, CH2), 1.55 (m, 4H, CH2), 2.27 (t, 2H, CH2CO, 3J ) 8.0 Hz), 2.60–2.72 (m, 3H, CH2), 2.96–3.11 (m, 3H, CONHCH2 and CH2), 3.14–3.28 (m, 2H), 3.37 (m, 1H, CH2), 3.75 (m, 2H, CHCON), 6.84 (s, broad, 1H, CONH), 7.35 (m, 9H, phenyl), 7.46 (m, 6H, phenyl). 13C NMR (CD3CN): 24.7, 26.6, 28.7, 28.9, 29.1, 31.3, 33.4, 35.7, 39.2, 46.4, 49.8, 50.7, 56.3, 56.8, 72.4 (NCH),

Mirtschink et al.

127.8 (d, 2JCP ) 8.0 Hz), 129.2, 134.1 (d, 3JCP ) 10.0 Hz), 144.6 (d, 1JCP ) 45.0 Hz), 168.3 (CONH), 174.4 (COOH). 31P NMR (CDCl3): 28.5 (RePPh3). ESI-MS (MeOH): m/z (%) ) 827 (100) for [C33H42N2O3PReS3]+. [12-({N,N-Bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino) dodecanoato](triphenylphosphine)rhenium(III) (ReIIb). Prepared according to the procedure for MeT4-Re-M0 under the use of T4 (48 mg, 0.1 mM), the hexathiourea Re(III) complex (77 mg, 0.1 mM) and M1 (52 mg, 0.2 mM) and an additional step for ester saponification. Column chromatography (silica gel 60, chloroform/ethyl acetate ) 2/1, Rf ) 0.45). Yield: 53 mg (60%) green solid. 1H NMR (CDCl3): 1.23–1.36 (m, 14H, CH2), 1.52–1.82 (m, 4H, CH2), 1.91 (m, 2H), 2.38 (t, 2H, CH2COOH, 3 J ) 6.0 Hz), 2.50 (m, 1H, CH2), 2.75–2.89 (m, 4H, CH2), 3.06–3.32 (m, 4H, CONHCH2 and CH2), 3.42 (m, 1H, CH2), 3.64 (m, 1H, CH2), 3.75 (m, 1H), 3.88 (m, 1H, CHCON), 5.89 (s, broad, 1H, CONH), 7.26–7.32 (m, 9H, phenyl), 7.47 (m, 6H, phenyl). 13C NMR (CDCl3): 25.0, 25.6, 25.7, 26.6, 27.0, 29.3, 29.4, 29.5, 29.7, 31.2, 32.9, 36.1, 40.1, 46.9, 49.7, 50.0, 51.0, 56.3, 73.1 (NCHCO), 127.9 (d, 2JCP ) 9.0 Hz), 129.1, 134.3 (d, 3JCP ) 11.0 Hz), 143.7 (d, 1JCP ) 46.0 Hz), 168.9 (CONH), 179.8 (COOH). 31P NMR (CDCl3): 28.1 (RePPh3). ESI-MS (MeOH): m/z (%) ) 883 (95) for [C37H50N2O3PReS3]+. [Methyl15-({N,N-Bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino)pentadecanoate](triphenylphosphine)rhenium(III) (ReMeIIc). Prepared according to the procedure for MeT4-Re-M0 under the use of T5 (26 mg, 0.05 mM), the hexathiourea Re(III) complex (38 mg, 0.05 mM) and M1 (26 mg, 0.1 mM). Column chromatography (silica gel 60, chloroform/ethyl acetate ) 5/4, Rf ) 0.45). Yield: 30 mg (65%) green solid. Crystals are available from acetonitrile solution at 4 °C. 1H NMR (CDCl3): 1.20–1.29 (m, 20H, CH2), 1.49–1.61 (m, 4H, CH2), 2.28 (t, 2H, CH2CO, 3J ) 8.0 Hz), 2.49 (m, 1H, CH2), 2.76–2.91 (m, 4H, CH2), 3.08–3.31 (m, 4H, CONHCH2 and CH2), 3.41 (m, 1H, CH2), 3.64 (s, 3H, OCH3), 3.75 (m, 1H, CHCON), 5.83 (s, broad, 1H, CONH), 7.27–7.34 (m, 9H, phenyl), 7.47 (m, 6H, phenyl). 13C NMR (CDCl3): 25.2, 27.7, 29.3, 29.4 (2C), 29.6, 29.7 (5C), 29.9, 34.5, 40.0, 46.9, 47.0, 49.8, 50.9, 51.7, 56.3, 73.1 (NCH), 127.9 (d, 2JCP ) 10.1 Hz), 129.8, 134.4 (d, 3JCP ) 11.1 Hz), 143.7 (d, 1JCP ) 46.3 Hz), 168.9 (CONH), 174.7 (COOMe). 31P NMR (CDCl3): 28.2 (RePPh3). ESI-MS (MeOH): m/z (%) ) 940 (90) for [C41H59N2O3PReS3]+. [15-({N,N-Bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino)pentadecanoic acid](triphenylphosphine)rhenium(III) (ReIIc). The ester function was saponified by addition of 0.5 mL 1 N sodium hydroxide to a solution of 1 mg of (ReMeIIc) in 2 mL dioxane kept at 75 °C for 10 min After acidification with 1 N hydrochloric acid to pH 3–4, the FA complex (ReIIc) was purified by semipreparative HPLC (Hypersil RP-18; eluent, acetonitrile/water/0.1% TFA; gradient, in 10 min from 50 f 100% acetonitrile; flow rate, 2 mL/min; retention time, 16.2 min ESI-MS (MeOH), m/z (%) ) 926 (80) for [C40H57N2O3PReS3]+. [12-({N,N-Bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino)dodecanoic acid](ethyldiphenylphosphinite-KP)rhenium (III) (ReIIIb). Prepared according to the procedure for MeT4Re-M0 by use of T4 (24 mg, 0.05 mM), the hexathiourea Re(III) complex (38 mg, 0.05 mM) and M5 (23 mg, 0.1 mM). Column chromatography (silica gel 60, tert-butyl methyl ether/hexane ) 3/1, Rf ) 0.75). Yield: 8 mg 19% of a dark green solid. 31P NMR (CDCl3): 16.5 (RePPh2OEt). UV/vis (CHCl3): λmax (nm) 444.5. ESI-MS (MeOH): m/z (%) ) 875 (100) for [C33H50NaN2O4PReS3]+. ESI-MS negative mode (MeOH): m/z (%) ) 851 (100) for [C33H50N2O4PReS3]-.


[12-({N,N-Bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino)dodecanoic acid][4-(isocyano-KC)-N-octylbutanamide]rhenium(III) (ReIVa). The precursor Re-dimethylphenylphosphine complex (MeT4-Re-M0) (12 mg, 0.0155 mM) was stirred with 4-isocyano -N-octylbutanamide (M9) (7 mg, 0.031 mM) for 2 h at room temperature in chloroform. After an additional step for ester saponification and removal of the solvent, the crude product was purified by column chromatography (silica gel 60, chloroform/ethyl acetate ) 5/4, +0.1% TFA, Rf ) 0.35). Yield: 12 mg (93%) green solid. 1H NMR (CDCl3): 0.85 (t, 3H, CH3, 3J ) 6.8 Hz), 1.19–1.35 (m, 24H, CH2), 1.46 (m, 2H, CH2), 1.55 (m, 4H, CH2), 2.09 (m, 2H, CH2), 2.27 (t, 2H, CH2CO, 3J ) 7.4 Hz), 2.39–2.45 (m, 1H, CH2), 2.51 (t, 2H, CH2CO, 3J ) 7.0 Hz), 2.78–2.91 (m, 2H, CH2), 2.98 (m, 1H, CH2), 3.09–3.42 (m, 9H), 3.49 (m, 1H, CH2), 3.88 (m, 1H, NCH), 4.70 (t, 2H, CNCH2, 3J ) 5.8 Hz), 6.02 (t, 1H, CONH, 3J ) 5.6 Hz), 6.41 (t, 1H, CONH, 3J ) 5.3 Hz). 13C NMR (CDCl3): 14.3, 22.9, 24.9, 27.1, 27.2, 29.0, 29.2, 29.3, 29.4 (2C), 29.5 (3C), 29.6, 29.7, 32.0, 32.1, 33.8, 34.1, 39.9, 40.2, 44.2, 44.8, 46.7, 48.0, 55.4, 71.7 (NCH), 166.4 (CN), 168.5 (CONH), 172.6 (CONH), 178.3 (COOH). ESI-MS (MeOH): m/z (%) ) 868 (80) for [C32H59N4NaO4ReS3]+. 99g Tc [12-({N,N-bis[2-(mercapto-KS)ethyl]cysteinyl-K2N2,S3}amino)dodecanoato][octyl(isocyano-KC)acetate]technetium(III) (Vc). The “carrier added” 99gTc reference complex was prepared according to the 99mTc complex synthesis by addition of 20 µL of a 10-6 M Na99gTcO4 solution to the 0.5 mL generator eluate. The complex was purified via semipreparative HPLC (Hypersil RP-18; eluent, acetonitrile/water/0.1% TFA; gradient, in 10 min from 50 f 100% acetonitrile; flow rate, 2 mL/min; retention time, 15.4 min). ESI-MS (MeOH): m/z (%) ) 732 (100) for [C30H55N3O5S3Tc]+. {N,N-Bis[2-(mercapto-KS)ethyl]cysteinato(3-)-K2N,S3}[methyl 15-(isocyano-KC)pentadecanoate]rhenium(III) (ReMeVIb). A further “4 + 1” NS3-Re-triphenylphosphine derivative (T2-ReM1) described in ref (18) (13 mg, 0.019 mM) was used as precursor in ligand exchange reactions with methyl 5-{[6(isocyano-κC)hexyl]thio} pentanoate (M16-Me) (8 mg, 0.03 mM). Column chromatography (silica gel 60, chloroform/ethyl acetate ) 1/1, Rf ) 0.65). Yield: 6.7 mg (59%) green solid. 1H NMR (CDCl3): 1.20–1.35 (m, 20H, CH2), 1.51–1.61 (m, 2H, CH2), 1.73–1.80 (m, 2H, CH2), 2.28 (t, 2H, CH2COO, 3J ) 7.6 Hz), 2.41–2.48 (m, 1H, CH2), 2.63–2.70 (m, 1H, CH2), 2.84–2.98 (m, 3H, CONHCH2 and CH2), 3.14–3.20 (m, 1H), 3.25–3.29 (m, 1H), 3.35–3.40 (m, 1H), 3.45–3.49 (m, 1H, CH2), 3.53–5.58 (m, 1H), 3.66 (s, 3H, OCH3), 3.73–3.77 (m, 1H, NCHCON), 4.75 (t, 2H, 3J ) 6.4 Hz, CNCH2). 13C NMR (CDCl3): 25.1, 26.9, 29.2, 29.4, 29.5, 29.7 (2C), 29.8 (2C), 29.9, 33.3, 34.4, 44.4, 45.3, 46.6, 48.2, 51.9, 55.1, 56.4, 69.3 (NCH), 163.8 (CNCH2), 175.2 (COOMe). ESI-MS (MeOH): m/z (%) ) 705 (90) for [C24H43N2O4ReS3]+. {N,N-bis[2-(mercapto-KS)ethyl]cysteinato(3-)-K2N,S3}[15-(isocyanoKC)pentadecanoic acid] rhenium(III) (ReVIb). The ester function was saponified by the addition of 0.5 mL 1 N sodium hydroxide to a solution of 1 mg of ReMeVIb in 2 mL dioxane kept at 75 °C for 10 min After acidification with 1 N hydrochloric acid to pH 3–4, the FA was purified by semipreparative HPLC (Hypersil RP-18; eluent, acetonitrile/water/ 0.1% TFA; gradient, in 10 min from 50 f 100% acetonitrile; flow rate, 2 mL/min; retention time, 13.0 min). ESI-MS (MeOH): m/z (%) ) 691 (90) for [C23H41N2O4ReS3]+. {N,N-Bis[2-(mercapto-KS)ethyl]cysteinato(3-)-K2N,S3}[methyl 5-{[6-(isocyano-KC)hexyl]thio} pentanoate]rhenium(III) (ReMeVIc). The “4 + 1” NS3-Re-triphenylphosphine derivative (T2-Re-M1) described in ref (18) (15 mg, 0.022 mM) was used as the precursor in a ligand exchange reactions with methyl 15isocyanopentadecanoate (M16-Me) (11 mg, 0.04 mM). Column


chromatography (silica gel 60, chloroform/ethyl acetate ) 1/1, Rf ) 0.55). Yield: 10 mg (65%) green solid. 1H NMR (CDCl3): 1.18–1.27 (m, 2H, CH2), 1.41–1.47 (m, 2H, CH2), 1.57–1.81 (m, 8H, CH2), 2.32 (t, 2H, CH2COO, 3J ) 7.6 Hz), 2.45 (m, 1H, CH2), 2.48–2.52 (m, 4H, CH2SCH2), 2.63–2.70 (m, 1H, CH2), 2.82–2.98 (m, 2H), 3.14–3.20 (m, 1H), 3.25–3.29 (m, 1H), 3.35–3.42 (m, 1H), 3.45–3.49 (m, 1H, CH2), 3.54–5.57 (m, 1H), 3.65 (s, 3H, OCH3), 3.73–3.77 (m, 2H, NCHCON + CH2), 4.76 (t, 2H, CNCH2, 3J ) 8.0 Hz). 13C NMR (CDCl3): 24.4, 26.5, 28.5, 29.3, 29.8, 29.9, 31.9, 32.3, 33.1, 33.9, 44.4, 45.2, 46.5, 48.2, 51.8, 55.1, 56.4, 69.6 (NCH), 161.6 (CNCH2), 174.3 (COOMe). ESI-MS (MeOH): m/z (%) ) 594 (90) for [C20H35N2O4ReS4]+. {N,N-Bis[2-(mercapto-KS)ethyl]cysteinato(3-)-K2N,S3}[5-{[6(isocyano-KC)hexyl]thio}pentanoic acid]rhenium(III) (ReVIc). The ester function was saponified by addition of 0.5 mL 1 N sodium hydroxide to a solution of 1 mg of (ReMeVIc) in 2 mL dioxane kept at 75 °C for 10 min After acidification with 1 N hydrochloric acid to pH 3–4, the FA was purified per semipreparative HPLC (Hypersil RP-18; eluent, acetonitrile/ water/0.1% TFA; gradient, in 10 min from 50 f 100% acetonitrile; flow rate, 2 mL/min; retention time, 11.8 min). ESIMS (MeOH): m/z (%) ) 580 (90) for [C19H33N2O4ReS4]+. (12-{[(Diphenylphosphino-KP)acetyl]amino}dodecanoic acid) [2,2′,2′′-(nitrilo-KN)triethane thiolato-(3-)-KS]rhenium(III) (T1-Re-M6 ) ReVIIa). 28 mg (0.97 mM) of the oxalic acid salt of tris(2-mercaptoethyl)amine (T1) and 100 mg (1.9 mM) of the crude monodentate 12-{[(diphenylphosphino)acetyl]amino}dodecanoic acid (M6) were refluxed for 3 h in methanol with 35 mg (0.046 mM) hexathiourea Re(III) complex {Re[SC(NH2)2]6}Cl3 × 1H2O as an appropriate Re(III) precursor. After removal of the solvent, the crude product was purified by column chromatography (silica gel 60, chloroform/ethyl acetate ) 4/1, Rf ) 0.50). The acidic reaction conditions in methanol lead to the corresponding methyl dodecanoato FA complex. (NMR data of the methyl ester Re complex are shown in Supporting Information.) For the saponification of the ester function, 1 mL 1 N aqueous sodium hydroxide was added to the dissolved methyl ester complex in 5 mL dioxane and stirred for 24 h. After acidification with TFA to pH 3–4 and evaporation of the solvent, a 3-fold extraction with chloroform/water followed. The united organic phases were dried with MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (silica gel 60, chloroform/ethyl acetate ) 9/1, Rf ) 0.5). Yield: 23 mg (58%). Mp > 220 °C (decomp.). 1H NMR (CDCl3): 1.05–1.30 (m, 16H), 1.58–1.63 (m, 2H), 2.33 (t, 2H, 3J ) 7.4 Hz), 2.60–3.20 (m, 12H), 3.0 (q, 2H, 3J ) 6.5 Hz), 3.75 (d, 2H, PCH2CONH, 2JHP ) 3.4 Hz), 6.2 (s, broad, 1H, CONH), 7.28 (t, 2H, phenyl, 3J ) 7.1 Hz), 7.36 (t, 4H, phenyl, 3J ) 7.4 Hz), 7.67 (dd, 4H, 3JHH ) 7.6 Hz, 3JHP ) 9.0 Hz). 13C NMR (CDCl3): 24.9, 27.0, 29.0, 29.1, 29.2, 29.4, 29.4, 29.4, 29.5, 33.9, 40.1 (CH2NHCO), 47.0, 48.0 (d, PCH2CONH, 1JCP ) 19.7 Hz), 60.4, 128.1 (d, 3JCP ) 9.5 Hz), 129.4, 133.0 (d, 2JCP ) 11.4 Hz), 144.4 (d, 1JCP ) 44.8 Hz), 168.5 (CONH), 177.2 (COO). 31P NMR (CDCl3): 17.0. ESI-MS (MeOH): m/z (%) ) 845 (70) [C32H48N2NaO3PReS3]+. X-ray Data Collection and Processing. The X-ray data were collected at room temperature (293 or 273 K) on a SMARTCCD diffractometer (Siemens), using graphite monochromatized Mo KR radiation (λ ) 0.710 73 Å). A summary of the crystallographic data of ReMeIIc is given in Table 2. The structures were solved by direct methods. After anisotropic refinement of the nonhydrogen atoms, the hydrogen positions were calculated according to ideal geometries. Empirical absorption corrections were made. Most of the calculations were carried out in the SHELXTL system with some local modifications. Selected bond lengths and angles are reported in figure


Mirtschink et al.

Table 1. Composition and Structures of Investigated Tc Compounds I–VII

legends of the crystal structures. CCDC 638660 (ReMeIIc) contains the supplementary crystallographic data for this paper. These files can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_request@ccdc. cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax +44 1223 336033. Synthesis Procedure for the Modified “4 + 1” 99mTc Complexes.99mTc-[12-({N,N-bis[2-(mercapto-KS)ethyl]cysteinylK2N2,S3}amino)dodecanoic Acid][4-(isocyano-KC)-N-octylbutanamide]technetium(III) (IVa). Before usage, all isocyano ligands were transferred to their corresponding Cu(I) complexes by addition of 0.25 equiv of Cu(I) chloride. 0.5 mL pertechnetate solution (100–500 MBq generator eluate) were added to a prepared EDTA kit containing EDTA (1 mg) and stannous (II) chloride (0.08 mg), as well as mannitol (5 mg). After 20 min tempering at 37 °C, a purity control was followed by radio TLC (silicagel/acetone). The 99mTc(III)–EDTA complex resulted with over 99% radiochemical purity. The aqueous EDTA-complex solution was added to a solution of the tetradentate ligand TX (0.5 mg) in 0.1 mL t-butyl alcohol {exemplary: 1-[(11-carboxyundecyl)amino]-3-mercapto-N,Nbis(2-mercaptoethyl)-1-oxopropan-2-aminium chloride (T4)},

and the monodentate phosphine or isocyano ligand MY (0.05 mg) in 0.2 mL n-propyl alcohol (exemplary: Cu(I) complex of 4-isocyano-N-octylbutanamide M9), and heated 30 min at 65 °C. After cooling to room temperature, the 99mTc–FA complex was purified via semipreparative HPLC (Hypersil RP-18; eluent, acetonitrile/water/0.1% TFA; gradient, in 10 min from 50 f 100% acetonitrile; flow rate, 2 mL/min (retention time for IVa, 14.6 min). For each compound group, at least one representative was identified as the expected 99mTc-FA by comparison of retention times with the nonradioactive Re reference FA complex. After the removal of acetonitrile by vacuum evaporation, 200 µL propylene glycol was added. The radiochemical yield of I–VII was 50–80%. Animals. All experiments were carried out on Wistar rats of both gender (body weight 200–250 g) purchased by Charles River (Sulzfeld, Germany). The investigations conformed to the Guide for the Care and Use of Laboratory Animals issued by the U.S. National Institutes of Health and were approved by the local government authority (AZ 24-9168.24-1-2002-14, AZ 24-9168.21-4/2004-1). Animals were kept under a 12 h light– dark cycle and fed with commercial animal diet and water ad libitum.

Synthesis and Evaluation of Technetium-Labeled Fatty Acids Table 2. Crystallographic Data for the X-ray Diffraction Study of Rhenium FA Complexes ReMeIIc ReMeIIc formula formula weight crystal system space group a [Å] b [Å] c [Å] R [°] β [°] γ [°] volume [Å 3] Z T [K] F [g/cm3] absorption coeff. [mm-1] F(000) λ (Mo KR) [Å] crystal size [mm3] θ-range from data collection index ranges reflections collected independent reflections goodness-of-fit on F2 R [I > 2σ (I)] R (all data)

C41H58N2O3PReS3 940.24 monoclinic P2(1)/n 19.80 (3) 9.719 (14) 23.21 (3) 90 109.46 (3) 90 4212 (11) 4 273 (2) 1.483 4.838 1920 0.71073 0.3 × 0.25 × 0.2 1.65–25.00 -23 e h e 12 -11 e k e 10 -27 e l e 27 21769 7402 1.097 R1 ) 0.1363 wR2 ) 0.2208 R1 ) 0.2328 wR2 ) 0.2552

Isolated Heart Preparation. Anesthesia was performed in two steps: After resting in a diethyl ether chamber for a short time, deep anesthesia was achieved by an intraperitoneal (i.p.) injection of 2–3 mL urethane (15%). In addition, 1000 U/kg i.p. heparin was injected. Hearts were rapidly removed during continuous cooling (Krebs-Henseleit buffer (KHB) 4 °C) and mounted within 1 min via the aorta vertically on a perfusion cannula. Perfusion was immediately started according to the Langendorff technique with a modified KHB containing (mM/ L): NaCl 116, KCl 4.6, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2.5, glucose 11, and pyruvate 2. The buffer was equilibrated with a 95% O2/5% CO2 gas mixture (resulting pH 7.38), FA free bovine albumin (BSA) (0.1%/14.9 µM/L), and maintained at 37 °C. The pulmonary artery was catheterized to permit anaerobic collection of the coronary venous effluent perfusate. Total coronary flow was measured using an ultrasonic transit-time flowmeter (T206 Transonic Systems Inc., Ithaca, NY) inserted in the arterial perfusion line and kept constant at 10 mL/min. Coronary perfusion pressure (CPP) was measured through a pressure transducer (Gould Statham, USA) connected to the perfusion cannula. Heart rate was calculated manually

Figure 3. Ventricular extraction (in % of infused activity) of modified 99m Tc-labeled FA evaluated with the Langendorff model.


from a CPP printout. “Arterial” and “venous” perfusate samples collected anaerobically were analyzed for pO2, pCO2, pH, HCO3- × (AVL 990S, AVL Scientific Corporation, Roswell, GA) before each intervention. Experimental Procedure. 99mTc-labeled FA were diluted in a ratio of 1:2 with KHB enriched with 6% BSA and left to equilibrate at room temperature for 30 min. After a period of 30 min to achieve hemodynamic stabilization of the beating heart, the 99mTc-labeled FA were infused proximal to the perfusion cannula in a ratio of 1:1000. Starting with the FA infusion, the complete venous effluent perfusate was collected in 12 consecutive samples of 15 s each (samples 1–12). Following the stop of radioactivity infusion, another four samples during sole KHB perfusion were collected (rinse out of possible coronary radiolabeled substances; samples 13–16). Released radioactivity (Aeff) was taken to equal the sum of the radioactivity recovered from these 16 effluent perfusate samples corrected for background activity. Then, perfusion was stopped, and hearts were quickly removed and separated into atria and right and left ventricles. For input calculation (Ain), 5 15 s perfusate samples were collected under identical radioactivity infusion and flow settings directly from the perfusion cannula in the absence of a heart. All perfusate and heart samples were measured with a gamma counter (Wallac 1470 Wizard, PerkinElmer Freiburg, Germany). Only experiments were used in which the calculated input (Ain) was in a range of (20% of the measured total radioactivity of perfusate plus heart tissue samples (Aeff + Atiss). Ventricular extraction (Ev) was calculated by dividing ventricular tissue radioactivity by the summed radioactivity of perfusate samples (Aeff) plus heart tissue samples (Atiss). Biodistribution Studies in Rats. Anesthesia was performed with an intraperitoneal injection (i.p.) of urethane (1.5 g/kg). A tail vein was surgically exposed, and a 200 µL (0.5 MBq) bolus of a solution containing the “4 + 1” 99mTc-FA compound and HSA (6%) in a 1:2 ratio were injected. The animals were sacrified by decapitation at 60 min after injection. The blood was immediately withdrawn by cardiac puncture with a heparinized syringe and the activity was counted. Organs were excised and weighed, and the radioactivity was determined (Wallac 1480 WizardTM gamma counter, PerkinElmer, Shelton, CT). The accumulated 99mTc activity in organs and tissues was calculated as percentage of the injected dose per organ (% ID/ organ) or per gram tissue (% ID/g tissue). Data are given as mean ( standard deviation (SD) of four animals each.

RESULTS Syntheses of FA Derivatives. With respect to the synthesis procedures, three important aspects deserve particular attention. First, the possibility to introduce the FA moiety to the tetradentate NS3 ligand results in more influentual capabilities for the Tc–FA complex properties by variation of the monodentate ligand. Functionalized isocyano ligands can be used as well as different commercially available phosphines or phosphites with varying chemical and sterical characteristics. Second, the design of isocyano-substituted active esters provides easy access to various isocyano compounds by multifarious functionalization reactions at the active ester group. Third, the application of the isocyanide compounds as Cu(I) complexes is a very important fact regarding enhancement of reactivity toward ligand exchange at the [Tc(FA-NS3)] core and concurrent stability of the isocyano group in the reaction medium. The advantages of this method are most obvious in all preparations in which the free FA appeared. As a consequence, the last saponification step, necessary when the isocyano FA is applied in the form of its ester, is redundant now, and thus, the synthesis proceeds more rapidly [50 min instead of 60 min; when

104 Bioconjugate Chem., Vol. 19, No. 1, 2008 Table 3. Biodistribution of Modified

99m

Mirtschink et al.

Tc “4 + 1” FA Derivatives in Ratsa

Ia time p.i. heart liver blood lungs kidneys heart/liver heart/blood heart/lungs

5 min 0.78 ( 0.08 11.83 ( 0.78 1.57 ( 0.02 1.21 ( 0.06 6.95 ( 0.51 0.07 0.50 0.64

time p.i. heart liver blood lungs kidneys heart/liver heart/blood heart/lungs

5 min 0.34 ( 0.04 4.96 ( 0.31 0.43 ( 0.05 0.59 ( 0.09 1.38 ( 0.18 0.07 0.79 0.58

time p.i. heart liver blood lungs kidneys heart/liver heart/blood heart/lungs

5 min 1.46 ( 0.70 13.38 ( 1.45 3.36 ( 1.10 2.00 ( 0.57 5.60 ( 0.88 0.11 0.40 0.73

IIa 60 min 0.33 ( 0.02 5.51 ( 0.99 0.62 ( 0.01 0.47 ( 0.02 2.59 ( 0.36 0.06 0.53 0.70

5 min 2.88 ( 0.19 14.14 ( 0.52 0.51 ( 0.08 1.04 ( 0.14 3.65 ( 0.22 0.20 5.65 2.77

60 min 0.04 ( 0.02 0.29 ( 0.08 0.10 ( 0.04 0.14 ( 0.04 0.30 ( 0.13 0.14 0.40 0.29

5 min 0.30 ( 0.03 12.45 ( 0.92 0.56 ( 0.17 0.85 ( 0.19 2.27 ( 0.42 0.02 0.54 0.35

Vc

5 min 0.96 ( 0.14 9.71 ( 1.39 2.53 ( 0.52 2.96 ( 0.31 0.91 ( 0.06 0.10 0.38 0.32

60 min 0.05 ( 0.01 2.47 ( 1.10 0.13 ( 0.03 0.38 ( 0.21 0.68 ( 0.23 0.02 0.38 0.13

5 min 0.58 ( 0.18 5.10 ( 0.77 0.27 ( 0.02 0.66 ( 0.26 0.98 ( 0.25 0.11 2.15 0.88

60 min 0.17 ( 0.03 0.60 ( 0.07 0.40 ( 0.06 0.39 ( 0.11 0.71 ( 0.16 0.28 0.43 0.44

5 min 1.04 ( 0.17 7.39 ( 0.56 0.66 ( 0.11 1.87 ( 0.37 3.04 ( 0.48 0.14 1.58 0.56

Vd

VIc

a

IIb 60 min 1.12 ( 0.22 17.14 ( 0.59 0.24 ( 0.01 0.39 ( 0.03 1.57 ( 0.13 0.07 4.67 2.87

IVa

VId 60 min 0.29 ( 0.09 0.88 ( 0.39 0.40 ( 0.06 0.83 ( 0.54 1.06 ( 0.18 0.33 0.45 0.35

5 min 1.15 ( 0.30 8.31 ( 0.78 3.36 ( 1.10 2.11 ( 1.21 6.79 ( 1.63 0.14 0.34 0.55

60 min 0.87 ( 0.07 7.62 ( 0.55 0.43 ( 0.07 2.00 ( 0.33 0.91 ( 0.07 0.11 2.02 0.49

60 min 0.38 ( 0.02 5.13 ( 0.30 0.09 ( 0.00 0.36 ( 0.05 0.91 ( 0.11 0.07 4.22 1.06 VII 60 min 0.27 ( 0.03 5.94 ( 0.61 0.22 ( 0.03 0.54 ( 0.02 0.84 ( 0.07 0.05 1.23 0.50

MW ( SD expressed as % ID/g organ, n ) 4 per time point.

comparing the reaction conditions for the nca procedure given in ref (14)]. A further positive aspect is the decreased reaction temperature (from 90 to 65 °C), which causes no drop in the yields (in some cases, the yields are even higher) and diminishes the risk of forming the corresponding Tc(I) hexaisocyanide derivatives as byproducts. The reaction route for the synthesis of “4 + 1” 99mTc/Re FA complexes with monodentate ligands bearing different metabolically stable functional groups is demonstrated in Figure 1. Experiments on the Isolated Perfused Rat Heart. Figure 3 shows the extraction results of all synthesized and investigated compounds (the complex composition of each FA derivative in addition to uptake values is presented in Table 2 in the Supporting Information). The length of the FA chain in the tetradentate ligand varied among 1, 8, 12, and 15 carbon atoms. The highest value of ventricular extraction in the isolated perfused Langendorff rat heart was achieved with the dodecanoic FA derivative Ib with 21.25 ( 1.28% ID. Four other compounds (IIa, IIb, IIc, and Vc) also exceeded the extraction value of 123 I-IPPA, for which a ventricular extraction of 15.51% ID was measured (14). The lowest extraction (0.6–2.09% ID) showed the “4 + 1” 99mTc-FA with a tetradentate ligand bearing only a carboxylic group (VIa–c). Biodistribution Studies in Rats. Table 3 shows the biodistribution of modified 99mTc “4 + 1” labeled FA in Wistar rats. Rapid clearance from blood was observed for all investigated compounds. The highest uptake in heart was achieved for IIa (2.88 ( 0.19% ID/g) with a remarkably high heart/blood ratio of 5.65 at 5 min postinjection (p.i.). Vc showed the most rapid washout from liver. Here, the initial liver uptake of 4.96 ( 0.31% ID/g was reduced down to 0.29 ( 0.08% ID/g at 60 min p.i. The elimination of compounds from group I–V proceeded predominantly via the hepatobiliary system, whereas VIc and

VId were excreted via the kidneys into the urine. The brain and the thyroid gland were almost free from radioactive species.

DISCUSSION The development of 99mTc-labeled FA, suitable for myocardial metabolism imaging is a challenging task. In an effort to synthesize 99mTc-“4 + 1” FA compounds with an improved liver clearance and high myocardial uptake rate, the concept of simple linear molecules with an end-positioned chelate was abandoned for more complex molecules with the chelate unit within the chain. This structure modification is enabled by a carboxylic group at the chelate backbone that allows binding of the FA moiety by an ω-amino group via the carboxylic acid amide bound. Thus, the remaining free coordination site on the tripodal metal core can additionally be functionalized by isocyanides or phosphorus(III) donors. The molecular structure of the triphenylphosphine complex ReMeIIc, the corresponding methyl ester of ReIIc, is presented as an example for the modified “4 + 1” FA species, bearing the FA unit bound to the tetradentate NS3 ligand. The compound crystallizes as a racemic structure in the monoclinic spacegroup P2(1)/n (see crystallographic data in Table 2). The centrosymmetry of the structure converts the 24(R) configuration into a 24(S) configuration. The structure of the chelate unit shows a large agreement with the basic structure of the unsubstituted “4 + 1” NS3-triphenylphosphine complex (19). The complex unit adopted a trigonal bipyramidal geometry where the three NS3 thiolate sulfurs form the trigonal plane and the amine nitrogen of the tetradentate FA ligand and the phosphorus atom of PPh3 occupy the apical positions (Figure 4). Figure 5 shows the linkage of the molecules via hydrogen bonds between the nitrogen of the amide and one oxygen of the carboxyl group, resulting in a head-to-tail arrangement. This interaction leads to the formation of chains running parallel to the b-axis.



Figure 4. Stereo drawing of the “4 + 1” FA-Re-triphenylphosphine complex with selected bond lengths (pm) and angels (deg) Re-P 230.0(5), Re-N1 221.7(17), Re-S1 222.5(6), Re-S2 222.2(6), Re-S3 221.9(6), and N1-Re-P 178.0(5).

These structures allow variations on both parts of the chain by introducing metabolically active groups (e.g., ester or amide) and, thus, the tuning of the breakdown properties to vary the clearance characteristics, especially from the liver. The potentially improved recognition by membranous and cellular FA transport and degradation enzymes would be an additional advantage of the modified “4 + 1” compounds. The chelate is positioned relatively far away from both the lipophilic tail section and the polar acid section, and on both endings of the FA compound, only inartificial groups were found. The configuration of natural FA was approximated by transferring the chelate from the end into the interior of the compound combined with a structural variation of the lipophilic tail composition. With the end-positioned carboxyl group, the synthesized derivates have a polar head serving as an important recognition feature. This polar section is so firmly connected with the chelate unit that decomposition is exclusively possible via β-oxidation. A lipophilic tail was constructed at the monodentate ligand, which represents a crucial characteristic of FA. Due to the included functional groups being cleaved in liver with a rate sequence of ester > ether > amide, the rapid formation of polar metabolites allows a fast washout from the liver (Figure 7). Within this structure, the position of the chelate was varied and moved from the carboxyl group to the lipophilic tail. Therefore, the influence on heart uptake and biodistribution of the total length of the resulting molecule and the relation of the polar section to the lipophilic section were studied.

Regarding the results obtained from the isolated heart model, some of the newly synthesized compounds achieved extraction results of about 20% ID. The common characteristics of these well-extracted compounds are the high level of lipophilicity and a FA chain length between 12 and 15 carbon atoms. Thus, functional groups with particular lipophilic properties incorporated in the tail section are an important feature to increase the myocardial uptake. However, the length of the tail section and the chelate position have a minor influence. In contrast, a shortening of the alkyl chain down to the carboxylic group as done in Vg or Vh clearly decreases the myocardial uptake in the Langendorff heart model from 18.01% ID as achieved with Vc down to 3.51% and 3.77% ID, respectively. The room-filling “4 + 1” 99mTc-chelate reflecting a dodecanoic FA derivative with respect to elongation, comparable with palmitic acid, may explain the preference for the mentioned FA chain length. Regarding the monodentate ligands used, isocyanides are preferred in consideration of the structural simplicity. However, one should keep in mind that the encumbering phosphine moiety is a part of many myocardium affine Tc tracers such as 99mTctetrofosmin or 99mTc-bis(diphosphines). Thus, it is not only permissive to involve phosphorus donor ligands in the intended tracer design, but rather reasonable profit may result from the binding properties of the phosphorus to technetium and the structural variability given by the three substituents. It involves the chance to locate positions at the phosphorus ligand that can be metabolized (as the phosphinite in complex Vb). Also worth mentioning is the prolonged myocardial retention time as


Mirtschink et al.

Figure 5. Molecules of the “4 + 1” FA-Re-triphenylphosphine complex building chains in the crystal structure. Dotted red lines indicate hydrogen bonds.

Figure 6. Lead structure (V) of the modified “4 + 1” 99mTc-FA concept for combining high myocardial uptake and fast liver clearance.

observed in biodistribution studies of VII, a “conventional” “4 + 1” FA derivative with an FA-bearing phosphine, and the triphenylphosphine containing compounds IIa and IIb. This fact is particularly obvious in comparison with “conventional” “4 + 1” FA with isocyano ligands (14). Initially, concerning in vivo stability of the presented 99mTc-FA compounds, metabolically stable (“inert”) monodentate ligands (e.g., phosphines and amides [groups II–IV]) were synthesized. In further course, better metabolizable ligands (e.g., ester [group V]) were developed. In this context, group IV compounds with an NHamide group incorporated into the tail can be regarded as precursors for derivatives developed later. At those compounds, the lipophilic tail is easily enzymatically cleaved by the introduction of an ester group (group V) (Figure 6). A good example of how structural changes in the monodentate ligand are reflected in biodistribution patterns is given by comparison of FA derivatives with the phosphino monodentate (group II) vs isocyanide/ester compounds (group V) (Figure 7). IIa fulfills the demand for high lipophilicity resulting in promising uptake values in the isolated perfused rat heart and in biodistribution (Table 2 and Figure 7). Furthermore, convenient heart/blood and heart/lung ratios at 5 min p.i. were found. Unfortunately, data on liver clearance were disappointing. Compared with the value after 5 min p.i., the measured concentration of radioactive species in the liver at 60 min p.i.

Figure 7. Biodistribution of 99mTc radioactivity concentration (% ID/g tissue) in male Wistar rats (n ) 4) at 5 min (A) and 60 min (B) after intravenous injection of 99mTc-labeled FA.

was even higher, indicating the formation of lipophilic metabolites. A similar high accumulation in the liver is described for unsubstituted “4 + 1” TcNS3–phosphine complexes (16).


In contrast, Vc exhibited accelerated liver clearance. Unfortunately, the myocardial uptake in biodistribution studies was rather low (Table 3). A possible reason for this shortcoming could be the untimely hydrolysis by plasma esterases. This is confirmed by the finding that the biodistribution of the dicarboxylic acid complexes VIb and VId shows a similar characteristic (low heart uptake and fast liver clearance). However, it should be considered that a more rapid rate of enzymatic hydrolysis in blood of rats (and mouse) is well-known. In addition, in case of the cationic technetium(I) hexakis (2carbomethoxy-2-isocyano propane)6+ (99mTc-CPI), metabolic products are less lipophilic and of negative charge, leading to a disabled myocardial uptake. In contrast, in species with decreased plasma hydrolysis activity such as guinea pigs, rabbits, and chickens, myocardial accumulation was clearly increased (20). Therefore, it is likely that ester hydrolysis in Vc by blood plasma enzymes may be slow enough for a high initial heart uptake in other animal species and in humans. In addition, evidence for a satisfactory heart uptake of Vc in the absence of rat plasma esterases is given in experiments with the isolated perfused rat heart model, where a ventricular extraction of 18.01% ID was obtained. For a better evaluation of this result, one should bear in mind that the myocardial extraction of 123IIPPA in the same experimental setup was 15.51% ID (14). A further attempt in the case of compounds VIa–VId was the combination of a tetradentate ligand containing the carboxyl group with a monodentate ligand bearing a free carboxyl group. It is well-known that the introduction of a carboxyl group in the NS3 ligand results in a distinct decrease of lipophilicity (16). Therefore, the extraction obtained in the isolated heart was low, no matter whether a carboxyl group containing isocyano ligand as in VIb or a phosphine as in VIa was used as a monodentate ligand. However, the renal excretion of the dicarboxylic acid derivatives VIb and VId was increased and the clearance from liver was accelerated. These results are in good agreement with the fast liver clearance of the ester containing compound Vc, which was metabolized by esterases to the corresponding dicarboxylic acid. As expected, the heart uptake obviously decreased in the isolated heart and to a lesser extent in biodistribution studies compared to the more promising results of the recently presented “conventional” “4 + 1” 99mTc-FA with unsubstituted tetradentate NS3 and isocyano FA ligands (14). In comparison with the recently presented 99mTc-cyclopentadienyltricarbonyltechnetium-pentadecanoic acid, [99mTc] CpTTPA (21), only IIa showed a similar myocardial uptake (2.88% ID/g). However, the heart/blood ratio at 5 min p.i. was clearly more promising for IIa (5.65 vs 2.93). Advantageous for the [99mTc] CpTT-PA is the combination of a satisfying uptake in heart and a rather low liver uptake. In conclusion, 29 modified 99mTc-“4 + 1” FA dedicated in groups I–VII according to their physicochemical properties were synthesized and evaluated using the isolated heart model and in vivo biodistributution studies. Differences in heart uptake and biodistribution were induced by varying the position of the Tc chelate, the FA chain length, the incorporation of various functional groups (amide, ether, ester) into the lipophilic tail, the insertion of blocking groups (thiol, phenylene, and alkine) in both the alkyl chain and the tail section, and the usage of two different kinds of monodentate ligands (isocyanides vs phosphines). Whereas some compounds show a high myocardial uptake, others are characterized by an accelerated washout from the liver. However, to date no compound has been found in which both properties are combined properly. Thus, the heart/ liver ratio could not be improved by the accomplished alterations of the binding mode between the “4 + 1” 99mTc chelate and the FA and the variations of the chelate position within the FA


chain. Although a clear structure/biobehavior relationship is still missing, nevertheless some important rules were objectified.

ACKNOWLEDGMENT This study was supported by an intramural grant (MedDrive programme 2003) funded by the Medical Faculty Carl Gustav Carus, by the DFG (Deutsche Forschungsgemeinschaft) grant SP 401, and by Nihon Medi-Physics Co., Inc., 9-8 Rokutanjicho, Nishinomiya-shi, Hyogo, 662-0918, Japan. The authors would like to thank Bianca Müller, Birgit Zatschler, and Regina Herrlich for excellent technical assistance. Supporting Information Available: Figure with used monodentate ligands, analytical data of benzyl-protected FA tristhiolate ligands, analytical data of precursors for isocyano ligands, analytical data of selected precursors of “4 + 1” Re reference compounds, and data for crystal structure. This material is available free of charge via the Internet at http://pubs.acs.org/ BC.

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