The [99mTc(N)(PNP)]2+ Metal Fragment: A Technetium-Nitrido

Bioconjugate Chem. 2003, 14, 1231−1242

1231

The [99mTc(N)(PNP)]2+ Metal Fragment: A Technetium-Nitrido Synthon for Use with Biologically Active Molecules. The N-(2-Methoxyphenyl)piperazyl-cysteine Analogues as Examples C. Bolzati,*,† A. Mahmood,‡ E. Malago`,§ L. Uccelli,§ A. Boschi,§ A. G. Jones,‡ F. Refosco,† A. Duatti,§ and F. Tisato† ICIS, CNR Corso Stati Uniti, 4, 35020 Padova, Italy, Harvard Medical School and Brigham and Woman’s Hospital, Boston, Massachusetts 02115, and Laboratory of Nuclear Medicine, Department of Clinical and Experimental Medicine, University of Ferrara, Via L. Borsari, 46, 44100 Ferrara, Italy. Received June 19, 2003; Revised Manuscript Received August 27, 2003

The incorporation of a bioactive molecule into a nitrido-containing 99mTc-complex has been successfully achieved by using the [TcN(PNP)]2+ metal fragment. In this strategy, the strong electrophilic [TcN(PNP)]2+ metal fragment efficiently reacts with bifunctional chelating ligands having a π-donor atom set, such as N-functionalized O,S-cysteine. The 2-methoxyphenylpiperazine (2-MPP) pharmacophore, which displays preferential affinity for 5HT1A receptors, was conjugated to the amino group of cysteine to obtain 2-MPPP-cys-OS, where 2-MPPP is 3-[4-(2-methoxyphenyl)piperazin-1-yl]propionate. The asymmetric Tc(V)-nitrido complexes, [99g/99mTc(N)(PNP)(2-MPPP-cys-OS)] (PNP ) PNP3, PNP4), were obtained in high yield (95%), by simultaneous addition of PNP and 2-MPPP-cys-OS ligand to a solution containing a starting 99g/99mTc-nitrido precursor. A mixture of syn and anti isomers was observed, the latter being the thermodynamically favored species. In vitro challenge experiments using the anti isomers with glutathione and cysteine indicated that no transchelation reaction occurs. Assessment of the in vitro 5HT1A receptor-affinity of the technetium complexes revealed that only the anti-PNP4 complex possesses some affinity for the receptor, but displayed negligible brain uptake in biodistribution studies in rats in vivo.

INTRODUCTION

One aspect of radiopharmaceutical development focuses on the design and synthesis of imaging agents that target specific receptors and transporters involved in certain pathological conditions. In vivo imaging via positron emission tomography (PET) provides the means to visualize receptor and transporter densities in living subjects and to estimate the inhibition of receptor, transporter density, and binding of endogenous (neurotransmitter) or exogenous (e.g. neuroleptic drug) ligands. This methodology is of general importance in elucidating how various neuropsychiatric conditions unfold in human subjects and for establishing the mechanism and efficacy of drug-treatment strategies. In this regard, investigation of the serotonergic system, which is implicated in several neuropsychiatric disorders including anxiety, depression, schizophrenia, and Alzheimer’s disease, has been a subject of particular interest. Recent reports have described [11C]WAY-100635 as a 11C-labeled radioligand that may provide a means to noninvasively image the 5HT1A receptors with PET (1, 2). While this agent is a potent and selective 5HT1A receptor antagonist, its initial high signal-to-noise ratio is compromised by the in vivo formation of small amounts of labeled metabolites that bind nonspecifically in various brain regions, thereby reducing image contrast. Thus the search for and devel* Author for correspondence. Phone +39 049 8295958. Fax +39 049 8702911. E-mail: [email protected]. † ICIS. ‡ Harvard Medical School and Brigham and Woman’s Hospital. § University of Ferrara.

opment of new 5HT1A radioligands is under active investigation (3, 4). Given the general availability in most nuclear medicine facilities of single-photon-emission tomography (SPECT) instrumentation compared with that for PET, the widespread availability of 99Mo-99mTc generators, and the near ideal imaging characteristics of 99mTc via SPECT, the potential utility of a technetium-99m-labeled receptor-specific agent for 5HT1A would be of interest. Thus, over the past few years various approaches have been proposed for synthesizing 99mTc-complexes capable of selectively binding central nervous system (CNS) 5HT1A receptors. Most of the reported complexes are based on the monooxo-Tc(V) core (5-11), with one utilizing the organometallic [99mTc(CO)3]+ metal fragment (12). Among all these strategies, however, the TctN core has yet to be developed for synthesizing 99mTc-labeled, receptortargeting radiopharmaceuticals. To explore the utility of the TctN core, we have developed a simplified route to incorporate biologically active molecules into stable 99mTcnitrido complexes. The strategy relies on the presence of asymmetric complexes containing two different polydentate ligands bound to the [TctN]2+ core (13). The synthesis entails the initial preparation of the substitution inert [Tc(N)(PXP)]2+ moiety, a strongly electrophilic fragment, that selectively reacts with bidentate chelating ligands (YZ) possessing π-donors as coordinating atoms, to form heterocomplexes of the type [Tc(N)(YZ)(PXP)]0/+ (14, 15); thus, for example, cysteine readily reacts as a bidentate ligand with the precursor complex [99mTc(N)(PXP)Cl2] via [O-, S-] or [NH2, S-] coordination in a nearly stoichiometric ratio, demonstrating that a terminal cys-

10.1021/bc034100g CCC: $25.00 © 2003 American Chemical Society Published on Web 10/18/2003

1232 Bioconjugate Chem., Vol. 14, No. 6, 2003

Figure 1. Diphospine (PNP) and bifunctional ligands utilized in this study.

teine group can act as an efficient chelating system for this metal fragment. The general requirements for a compound to cross the blood-brain barrier (BBB) in appreciable amounts must be met when designing complexes to target CNS receptors. These include that the complex be neutral in charge, lipophilic in character, and possess a molecular weight lower than 700 Da. Considering these criteria, we focused on di-negative [O-,S-], N-functionalized, cysteine-containing ligands, with the cysteine N-terminus conjugated via a propionate link to the biological molecule of interest, the arylpiperazine [(2-methoxyphenyl)piperazine], known to be active toward the 5HT1A subclass of serotonergic receptors. The final bidentate ligand (2-MPPP-cys-OS), when reacted with the [99mTc(N)(PXP)Cl2] fragment would thus provide TctN-based neutral, lipophilic complexes and a means of assessing the feasibility of this approach. Herein we report the application of this labeling procedure to the preparation of asymmetrical 99g/99mTcnitrido complexes with the diphosphine ligands PNP3 and PNP4 and the bidentate chelating ligand 2-MPPPcys-OS (Figure 1). EXPERIMENTAL PROCEDURES

Caution! 99gTc is a weak β- -emitter (Eβ- ) 0.292 MeV, t1/2 ) 1.12 × 105 years). All manipulations were carried out in laboratories approved for low-level radioactivity use. Handling milligram amounts of 99gTc does not present a serious health hazard since common laboratory glassware provides adequate shielding and all work is performed in approved and monitored hoods and gloveboxes. Bremsstrahlung is not a significant problem due to the low energy of the β-particles; however, proper radiation safety procedures must be followed at all times, and particular care should be taken when handling solid samples. General. All chemicals and reagents were purchased from Aldrich Chemicals. All solvents were reagent grade and were used without further purification. Due to the tendency of the diphosphine ligands to oxidize, all the solvents used in reactions with PNP3 and PNP4 were previously degassed to remove dissolved trace oxygen. Commercially available NH4[99gTcO4] (Oak Ridge National Laboratories) was purified from a black contaminant (99gTcO2 × nH2O) by addition of H2O2 and NH4OH solutions followed by recrystallization as NH4[99gTcO4] from hot water. Technetium-99m as Na[99mTcO4] was eluted from a 99Mo-99mTc generator provided by Nycomed Amersham-Sorin (Saluggia, Italy). [99gTc(N)Cl2(PPh3)2] was prepared as previously described (16). The diphosphine ligands bis(dimethoxypropylphosphinoethyl)methoxyethylamine, [PNP3 ) (CH3OC3H6)2P(CH2)2N(C2H4OCH3)(CH2)2P(C3H6OCH3)2] and bis(dimethylphosphinoethyl)methylamine [PNP4 ) (CH3)2P(CH2)2N(CH3)(CH2)2P(CH3)2] were purchased from Argus Chemicals (Prato, Italy). [3H]-8-Hydroxy-N,N-dipropyl-2-amino-

Bolzati et al.

tetralin ([3H]-8-OH-DPAT, specific activity ) 147.2 Ci/ mmol) and Aquensure were purchased from New England Nuclear Research Products (NEN, Boston, MA). Female Sprague-Dawley rats were acquired from Morini (Reggio Emilia, Italy). Elemental analyses (C, H, N, S) were performed on a Carlo Erba 1106 elemental analyzer. FT IR spectra were recorded on a Nicolet 510P Fourier transform spectrometer, in the range 4000-200 cm-1 and in KBr mixture using a Spectra-Tec diffuse-reflectance collector accessory. 1H, 13C, and 31P NMR spectra were collected on a Brucker 300 instrument, using SiMe4 as internal reference (1H and 13C) and 85% aqueous H3PO4 as external reference (31P). Chromatographic purification of the compounds was accomplished on silica gel columns: for the bifunctional ligand, 120 × 5 cm, 70-230 mesh (Aldrich) and for the asymmetrical 99gTc-complexes, 30 × 2 cm, 70-230 mesh (Merck). Thin-layer chromatography (TLC) (SiO2 F254S, Merck) and high performance liquid chromatography (HPLC) analyses were used to evaluate the radiochemical purity (RCP) and stability of the compounds. Radioactivity on TLC plates was detected and measured using a Cyclone instrument equipped with a phosphorus imaging screen and OptiQuant image analysis software (Packard, Meridian, CT). HPLC was performed on a Beckman System Gold instrument equipped with a programmable solvent Model 126, a sample injection valve 210A, a scanning detector Module 166, and a radioisotope detector Model 170. HPLC analysis was carried out on a reversed-phase Hamilton PRP-1 precolumn (45 × 4.1 mm), and a reversed-phase Hamilton PRP-1 column (250 × 4.1 mm, L ) 10 µm) using the chromatographic condition reported in Table 1; flow rate: 1 mL/min. Log k0′ of the complexes was determined by HPLC chromatography as reported previously (17). The results are summarized in Table 1. 3-[4-(2-Methoxyphenyl)piperazin-1-yl]propionic Acid Methyl Ester (1). 1-(2-Methoxyphenyl)piperazine hydrochloride (2-MPP) (4 g, 0.017 mol) and N,N-diisopropylethylamine (4.49 g, 0.035 mol) were dissolved in CH2Cl2 (150 mL). To this solution was added methyl 3-bromopropionate (1.947 g, 0.012 mol), and the reaction mixture was stirred at room-temperature overnight. The solvent was evaporated, and the residual oil was suspended in water (50 mL) and extracted with CH2Cl2 (50 mL × 3). The combined CH2Cl2 layers were reduced in volume, and the resulting pale yellow oil was purified by silica gel chromatography eluting with CH2Cl2 followed by a 3% MeOH/CH2Cl2 solution (3.51 g, 72%). 1H NMR (CDCl3) δ: 6.97-6.82 (4H, Ar), 3.82 (s 3H, OCH3), 3.66 (s, 3H, CH3), 3.06 (m, 4H, CH2), 2.76 (t, 2H, CH2CO), 2.65 (m, 4H, CH2), 2.54 (t, 2H, CH2). Anal. Calcd for C15H22N2O3: C, 64.73; H, 7.97; N 10.06; O, 17.24; found: C, 64.72; H, 7.79; N, 10.06; O, 17.02. 3-[4-(2-Methoxyphenyl)piperazin-1-yl)]propionate (2). Compound 1 (3.42 g, 12.3 mmol) was dissolved in MeOH (30 mL), and 10 mL of an aqueous solution containing KOH (1.034 g, 18.4 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated, and the residue, dissolved in the minimum amount of CH2Cl2, was purified by chromatography using a short silica gel column eluted with 20% MeOH/CH2Cl2 solution (3.01 g, 81%). 1H NMR (CDCl3) δ: 7.08-6.89 (4H, Ar), 3.89 (s, 3H, OCH3), 3.24 (m, 4H, CH2), 3.02 (m, 4H, CH2), 2.96 (t, 2H, CH2CO), 2.63 (t, 2H, CH2). Anal. Calcd for C14H19KN2O3: C, 55.60; H, 6.33; N, 9.26; O, 15.87; found: C, 55.02; H, 6.55; N, 9.31; O, 15.8.

Labeling of Bioactive Molecule with [99mTc(N)(PNP)]2+ Synthon Table 1. Chromatographic Data for

99g/99mTc

Bioconjugate Chem., Vol. 14, No. 6, 2003 1233

Complexes retention time (min)

complex [Tc(N)Cl2(PNP3)] [Tc(N)Cl2(PNP4)] anti-[Tc(N)(2-MPPP-cys-OS)(PNP3)] syn-[Tc(N)(2-MPPP-cys-OS)(PNP3)] anti-[Tc(N)(2-MPPP-cys-OS)(PNP4)] syn[Tc(N)(2-MPPP-cys-OS)(PNP4)] [Tc(N)(2-MPPP-cys-OS)2] [Tc(N)(OS-TA)(PNP4)] [Tc-HMPAO] [Tc-ECD]

Mw

Rf (SiO2)

99gTc

667.52 391.14 962.07 962.07 685.69 685.69 843.91

a

18.65c

0.0 0.0b 0.5a 0.4a 0.5b 0.4b 0.0a,b

22.01d 23.58c 23.02c 27.48d 26.37d -

99mTc

19.3 + 22.5 + mixd 23.86c 23.28c 27.82d 26.66d mixc,d 21.1d

% yields 99gTc

99mTc

Log P

Log k0′

48.5 43.6 38.6 33.6 -

89 9.6 73.6 7.6 -

2.42

4.84 4.84 3.28 3.28

ref

mixc

1.88

1.90 1.64

18 18

a TLC: MeOH (2% NH OH 20%)/CHCl (10/90). b TLC: CH Cl /MeOH/NH OH 20% (89/10/1). c HPLC: Reverse-phase C 4 3 2 2 4 18 column; A ) 5 mM DMGA, pH 7 (DMGA ) 3,3-dimethylglutaric acid), B ) CH3CN. Gradient: 0-5 min, B, 0%, 5-20 min, B, 70%, 25-30 min, B, 70%, 30-35 min, B, 0%. d Reverse-phase C18 column, A ) TFA 0.01 M, pH 3 (TFA ) trifluoroacetic acid), B ) CH3CN. Gradient 0-2 min, B, 0%; 2-23 min, B, 30%, 25-30 min, B, 70%, 30-35 min, B, 70%, 35-40 min, B, 0%.

2-Amino-3-tritylsulfanyl-propionic Acid Ethyl Ester (3). A suspension of (S)-trityl-L-cysteine (1.125 g, 3.1 mmol) and ethyl p-toluensulfonate (2.250 g, 6.2 mmol) in dry ethanol was refluxed under an argon atmosphere for 48 h, during which time dissolution occurred. After evaporation of the solvent, the residue was dissolved in 30 mL of an aqueous solution of KHCO3 (pH 8) and extracted three times with an equal volume of CH2Cl2. The combined organic phases were evaporated, and the pale yellow oil was redissolved in the minimum amount of CH2Cl2. Finally, Et2O was added to precipitate a white powder. The precipitate was separated by filtration, and the ether filtrate was evaporated to obtain the crude ester. Purification was achieved by SiO2 chromatography, eluting with a 2% MeOH/CH2Cl2 solution (0.97 g, 80%) 1 H NMR (CDCl3) δ: 7.46-7.23 (15H, Ar), 4.14 (q, 2H, CH2O), 3.22 (m, 1H, CHNH2), 2.59 (q, 1H, CH2S), 2.50(q, 1H, CH2S), 1.25 (t, 3H, CH3). Anal. Calcd for C24H25NO2S: C, 73.62; H, 6.44; N, 3.58; S, 8.19; found: C, 73.02; H, 6.96; N, 3.66; S, 8.06. 2-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propionylamino}-3-tritylsulfanylpropionic Acid Ethyl Ester (4). Compounds 2 (0.310 g, 1.15 mmol) and 3 (0.45 g, 1.15 mmol) were dissolved in CH2Cl2 (30 mL). The solution was cooled at -78 °C in a dry ice acetone bath, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (0.330 g, 1.73 mmol), previously dissolved in the minimum amount of DMF, was added. The reaction mixture was stirred at -78 °C, under argon atmosphere for 1 h, allowed to reach room temperature, and stirred overnight. The solvent was removed, and the residue was subjected to H2O/CH2Cl2 extraction. The combined CH2Cl2 layers were dried with anhydrous Na2SO4, evaporated to minimum volume, and purified by SiO2 chromatography eluting with 3% MeOH/CH2Cl2 solution (0.70 g, 96%). 1 H NMR (CDCl3) δ: 9.53 (d, 1H, NH), 7.36-7.18 (15H, Ar), 7.03-6.80 (4H, Ar), 4.73 (m, 1H, CHNH), 4.15 (t, 2H, CH2Npip), 3.87 (s, 3H, OCH3), 3.14 (m, 4H, CH2pip), 2.93 (m, 2H, CH2CO), 2.79 (m, 4H, CH2pip), 2.64 (q, 1H, CH2OC(O)), 2.53 (q, 1H, CH2OC(O)), 2.45 (d, 2H, CH2S), 1.25 (t, 3H, CH3). Anal. Calcd for C38H43N3O4S: C, 71.56; H, 6.79; N, 6.58; S, 5.03; found: C, 70.18; H, 6.80; N, 6.59; S, 5.03. 2-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propionylamino}-3-trityl-sulfanylpropionic Acid (5). Compound 4 (2.027 g, 0.0032 mol) was dissolved in MeOH (30 mL), and 5 mL of an aqueous solution containing KOH (0.267 g, 0.005 mol) was added. The reaction mixture was stirred at room temperature for 1 h. After the solvent was evaporated, the residue was redissolved in water, the pH was adjusted to 3 with HCl (2 M), and

the ligand was extracted with CH2Cl2 (30 mL × 3). The combined CH2Cl2 layers were reduced to a minimum volume with the pure hydrochloride salt precipitating as a white powder with the addition of anhydrous Et2O (1.61 g, 83%). The powder was dissolved in methanol and treated with KHCO3 to obtain the free base. 1H NMR (CD3COD) δ: 7.85 (s, 1H, NH), 7.42-7.21 (15H, Ar), 7.10-6.93 (4H, Ar), 4.44 (t, 1H, CHNH), 3.89 (s, 3H, OCH3), 3.37-3.26 (m, 12H, alkyl), 2.73-2.66 (m, 4H, alkyl). FT IR (cm-1): [3258 ν (NH)], [2930-2835 ν (CH2CH2)], [1750 ν (COOH)], [1683 ν (CONH)], [1240 ν (OCH3)], [743, 701 ν (CHdCH)]. Anal. Calcd for C36H39N3O4S‚2HCl: C, 63.38; H, 6.05; N, 6.15; S, 4.7; found: C, 63.46; H, 6.85; N, 6.31; S, 4.59. 2-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propionylamino}-3-sulfhydryl-propionic Acid (2-MPPPcys-OS) (6). Trityl deprotection of the thiol was performed by dissolving 5 (0.2 g, 0.293 mmol) in trifluoroacetic acid (TFA) (5 mL). The resulting yellow solution was stirred for 5 min, and then triethylsilyl hydride was added by drops until the solution became colorless. The solution was evaporated to dryness to remove TFA and subsequently dried under vacuum and used without further purification. Synthesis of [99gTc(N)Cl2(PNP3)] (7). To [Tc(N)Cl2(PPh3)2] (118 mg, 0.166 mmol) suspended in CH2Cl2 (10 mL) was added PNP3 (120 mg, 0.248 mmol) dissolved in CH2Cl2 (5 mL). The solution was stirred at reflux for 30 min. The initial orange-pink solution changed color to yellow. After the solution was cooled, the solvent was removed by a gentle stream of nitrogen and the oily residue was washed with Et2O, dissolved in the minimum amount of EtOH, and precipitated with n-hexane. The yellow powder was collected by filtration, washed with Et2O and n-hexane again, and dried under vacuum (94.04 mg, 85%). 1H NMR (CDCl3) δ: 3.44-1.63 (H, alkyl). 31P NMR (CDCl3) δ: 31.0 (broad singlet). FT IR (cm-1): [1054 ν (TctN)], [2979-2817 ν (C-H)]. Anal. Calcd for C23H51N2O5P2Cl2Tc: C, 41.38; H, 7.71; N, 4.21; found: C, 40.57; H, 7.87; N, 4.51. Synthesis of [99gTc(N)Cl2(PNP4)] (8). To [Tc(N)Cl2(PPh3)2] (90 mg, 0.130 mmol) suspended in CH2Cl2 (10 mL) was added PNP4 (30 mg, 0.145 mmol) dissolved in EtOH (5 mL). The solution was stirred at room temperature for 30 min during which time the initial orangepink solution changed color to yellow. The solvent was removed by a gentle stream of nitrogen, and the residue was washed with Et2O and treated with n-hexane. The precipitated yellow residue was redissolved in methanol, and a residual white powder was separated by filtration. The yellow filtrate was evaporated, and the residue was


treated with Et2O to provide a yellow precipitate, which was collected by filtration, washed with Et2O and nhexane, and dried under vacuum (40.67 mg, 80%). 1H NMR (CDCl3) δ: 2.47 (m, 4H, NCH2), 2.26 (m, 2H, PCH2endo), 2.11 (s, 3H, NCH3), 1.92 (m, 2H, PCH2exo), 2.02, 1.99, 1.78, 1.76 (4s, 12H, PCH3). 13C NMR (CDCl3) δ: 51.88 (NCH2), 46.20 (NCH3), 27.65, 27.45 (PCH2), 17.96, 17.76, 14.98, 14.76 (PCH3). 31P NMR (CDCl3) δ: 23.1 (bs). FT IR (cm-1): [1045 ν (TctN)], [2971-2804 ν (C-H)]. Anal. Calcd for C9H23N2Cl2P2Tc: C, 27.63; H, 5.90; N, 7.16; found: C, 27.01; H, 5.79; N, 7.01. Synthesis of [99gTc(N)(2-MPPP-cys-OS)(PNP3)] Anti (9a) and Syn (9b). Method A: Compound 7 (57.3 mg, 0.086 mmol) was dissolved in EtOH (5 mL) along with five drops of neat Et3N. To this solution was added 2-MPPP-cys-OS ligand (41 mg, 0.108 mmol) dissolved in CH2Cl2 (5 mL). The reaction mixture was stirred at reflux for 60 min. No apparent color change from yellow was observed during the reaction. After the solution was cooled, the solvent was removed, and the residue was washed with Et2O. The yellow oily residue was dissolved in the minimum amount of CHCl3 and chromatographed on a silica column, preconditioned with CHCl3, and eluted with a mixture of CHCl3/MeOH/NH4OH (20%) (95/5/3). Two yellow bands were separated and collected; the first fraction was identified (vide infra) as the pure anti isomer 9a (35%, based on Tc) and the second fraction as the pure syn isomer 9b (29%, based on Tc). Method B: To [99gTc(N)Cl2(PPh3)2] (29.32 mg, 0.041 mmol) suspended in CH2Cl2 (5 mL) were added PNP3 (20 mg, 0.043 mmol) dissolved in CH2Cl2 (5 mL), 2-MPPPcys-OS ligand (37.08 mg, 0.060 mmol) dissolved in CH2Cl2 (5 mL), and neat Et3N (five drops). Within 10 min the initial orange-pink solution turned yellow. The reaction mixture was further stirred at room temperature under a nitrogen atmosphere, for 1 h. The solvent was evaporated, and the crude yellow residue was dissolved in CHCl3 (1 mL) and chromatographed on silica. After the column was eluted with the mobile phase described above, two yellow bands were separated and isolated as yellow oily residues. The first fraction was identified as the anti isomer 9a (48.5%, based on Tc) and the second fraction as the syn isomer 9b (43.6%, based on Tc). These two isomers were identical to those prepared by method A, as confirmed by TLC, HPLC, and NMR data. Complex 9a was soluble in chlorinate solvents and in alcohols, and insoluble in diethyl ether and n-hexane. 1H NMR (CDCl3) δ: 8.04 (d, 1H, NHC(O)), 7.00-6.82 (4H, C6H4OCH3), 4.83 (m, 1H, CHNHC(O)), 3.85 (s, 3H, C6H4OCH3), 3.36-3.19 (5s, 15H, aliphatic OCH3), 3.32 and 2.41 (m, 2H, S-CH2exo and S-CH2endo), 3.16 and 2.74 (bs, 4H+4H, CH2pip), 3.55-1.55 (36H+4H, CH2 groups). 13C NMR (CDCl3) δ: 177.65, 170.75 (CdO), 152.28, 141.42, 122.75, 120.96, 118.39, 111.21 (C6H4OCH3), 73.14, 72.93, 72.63, 72.48, 68.34 (CH2O), 58.67, 58.57 (OCH3), 56.63 (SCH2CH), 55.37(C6H4OCH3), 54.13 (CH2Npip), 53.03 (CH2pip), 51.54 (NCH2CH2OCH3), 50.48 (CH2pip), 47.27, 46.13 (NCH2CH2P), 33.47, 29.70 (SCH2CH), 25.77, 24.77, 23.85, 22.80, 18.20, 16.27 (PCH2CH2CH2OCH3). 31P NMR (CDCl3) δ: 32.8 (broad s), 25.4 (broad s). FT IR (cm-1): [1050 ν (TctN)], [3300 ν (NH)], [2933-2817 ν (C-H)], [1732 ν (CO)O], [1636 ν NH(CO)], [1240 ν (OCH3)], [749 ν (b, CHdCH)]. Anal. Calcd for C40H74N5O9P2STc: C, 49.93; H, 7.75; N, 7.27; S, 3.34; found: C, 49.85; H, 7.8; N, 7.28; S, 3.32. Complex 9b was soluble in chlorinate solvents and alcohols and insoluble in diethyl ether and n-hexane. 1H NMR (CDCl3) δ: 8.26 (d, 1H, NHC(O)), 7.00-6.82 (4H, C6H4OCH3), 4.73 (m, 1H, CHNHC(O)), 3.86 (s, 3H, C6H4-

Bolzati et al.

OCH3), 3.36-3.20 (5s, 15H, aliphatic OCH3), 3.21 (m, 2H, SCH2CH), 3.14 and 2.76 (4H+4H, CH2pip), 3.60-1.55 (36H+4H, CH2 groups). 13C NMR (CDCl3) δ: 176.81, 171.15 (CdO), 152.01, 141.42, 122.68, 120.89, 118.27, 111.10 (C6H4-OCH3), 73.19, 73.01, 72.73, 72.33, 68.03, 58.67, 58.51, 56.56 (SCH2CH), 55.44 (C6H4OCH3), 53.99, 52.98, 51.34, 50.40, 47.24, 46.03, 33.42, 29.70 (SCH2CH), 25.60, 24.82, 23.65, 22.40, 17.97, 16.23. 31P NMR (CDCl3) δ: 34.6 (broad s), 27.1 (broad s). FT IR (cm-1): [1049 ν (TctN)], [3374 ν (NH)], [2949-2810 ν (CH)], [1660-1636 ν (CO)O, ν NH(CO)], [1240 ν (OCH3)], [749 ν (b, CHd CH)]. Anal. Calcd for C40H74N5O9P2STc: C, 49.93; H, 7.75; N, 7.27; S, 3.34; found: C, 49.7; H, 7.71; N, 7.3; S, 3.35. Synthesis of [99gTc(N)(2-MPPP-cys-OS)(PNP4)] Anti (10a) and Syn (10b). Method A: To compound 8 (33.56 mg, 0.086 mmol) dissolved in EtOH (5 mL) were added neat Et3N (five drops) and 2-MPPP-cys-OS ligand (39 mg, 0.103 mmol) dissolved in CH2Cl2 (5 mL). The mixture was stirred at reflux for 60 min. No color change from the initial yellow was observed throughout the reaction. After the solution was cooled, the solvent was removed by a gentle stream of nitrogen and the residue was washed with Et2O followed by n-hexane. The yellow oil was dissolved in the minimum amount of CH2Cl2 and loaded onto a silica column conditioned with CH2Cl2. The column was eluted with a mixture of CH2Cl2/MeOH/NH4OH (20%) (89/10/1), and two yellow bands were separated and collected. The eluate was evaporated, and the residues were treated with Et2O to precipitate the complexes. The first pale yellow product was identified as the pure anti isomer 10a (35%, based on Tc) and the second lemonyellow complex as the pure syn isomer 10b (19%, based on Tc). Method B: To [99gTc(N)Cl2(PPh3)2] (30.6 mg, 0.043 mmol) suspended in CH2Cl2 (5 mL) were added PNP4 (10 mg, 0.048 mmol) dissolved in CH2Cl2 (5 mL), 2-MPPPcys-OS ligand (38 mg, 0.062 mmol) dissolved in CH2Cl2 (5 mL), and an excess of Et3N (five drops). The reaction mixture was stirred at reflux for 1 h under nitrogen atmosphere. The initial orange-pink solution turned yellow. After the solution was cooled, the solvent was evaporated, and the resulting oily residue was dissolved in CH2Cl2 (1 mL) and chromatographed on a silica column using the conditions described above. Two fractions were collected, 10a (48.5%, based on Tc) and 10b (19.5%, based on Tc). These two isomers were identical to those prepared by method A, as confirmed by TLC, HPLC, and NMR data. Complex 10a was soluble in chlorinate solvents and alcohols and insoluble in diethyl ether. 1H NMR (CDCl3) δ: 8.33 (d, 1H, NH-C(O)), 6.98-6.83 (4H, C6H4OCH3), 4.89 (m, 1H, CHNHC(O)), 3.86 (s, 3H; C6H4OCH3), 3.34 (dd, 1H, SCH2exo), 2.47 (m, 1H; SCH2endo), 3.17 (bs, 4H, CH2pip), 2.78 (m 6H; CH2pip, CH2en), 2.47 (m, 2H, CH2en), 2.6-1.9 (8H, PCH2CH2N), 2.0 (s, 3H, NCH3), 1.78, 1.75, 1.72, 1.69 (4s, 12H, P-CH3). 13C NMR (CDCl3) δ: 177.95170.67 (CdO), 152.19, 141.29, 122.70, 120.88, 118.39, 111.10 (C6H4-OCH3), 56.92 (SCH2CH), 55.32 (OCH3), 54.13 (CH2N), 52.97, 50.36 (CH2pip); 51.80, 51.19 (PCH2CH2N), 45.05 (NCH3), 33.28 (C(O)CH2CH2N), 30.13 (SCH2CH), 28.27 (d), 26.19 (d, P-CH2), 19.13 (d), 15.46 (d), 13.55 (d), 11.75 (d, P-CH3). 31P NMR (CDCl3) δ: 20.0 (broad s), 14.0 (broad s). FT IR (cm-1): [1047 ν (TctN)], [3296 ν (NH)], [2936-2826 ν (-CH)], [1700-1636 ν (CO)O, ν NH(CO)], [1240 ν (OCH3)], [747, 726 ν (b, CHdCH)]. Anal. Calcd for C26H46N5O4P2STc: C, 45.55; H, 6.76; N, 10.22; S, 4.68; found: C, 45.2; H, 6.78; N, 10.18; S, 4.71. Complex 10b was soluble in chlorinate solvents and alcohols and insoluble in diethyl ether. 1H NMR (CDCl3)

Labeling of Bioactive Molecule with [99mTc(N)(PNP)]2+ Synthon


Table 2. pH and Temperature Dependence of Syn f Anti Isomerization for Complex syn-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] (10b) % of syn-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] (10b) buffer

pH

T, °C

0

12 h

24 h

3d

5d

7d

Ac-/HAc Ac-/HAc Ac-/HAc H2PO4-/HPO42H2PO4-/HPO42H2PO4-/HPO42HCO3-/CO32HCO3-/CO32HCO3-/CO32water water water

5.0 5.0 5.0 7.5 7.5 7.5 9.0 9.0 9.0 -

4 25 37 4 25 37 4 25 37 4 25 37

92.0 92.1 92.0 93.5 93.5 93.5 91.0 91.0 91.0 93.0 93.0 93.0

86.0 81.1 77.2 87.0 82.1 77.2 85.0 80.1 75.2 87.0 82.1 77.2

82.3 79.6 65.3 83.3 80.6 66.5 81.3 78.6 66.3 83.3 80.6 66.3

77.6 72.2 47.2.2 77.7 72.0 45.0 76.7 72.2 44.0 77.7 72.0 45.2

71.4 65.8 21.3 73.4 65.2 23.3 73.4 65.4 20.1 73.4 65.2 20.3

69.7 55.5 10.8 71.2 54.5 11.6 71.2 54.3 10.9 71.2 54.5 10.6

% of syn-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] (10b)

water water

-

55 100

0

15 min

60 min

120 min

6h

24 h

90.0 90.0

87.2 11.5

83.3 11.6

71.3 12.0

39.7 -

10.2 -

δ: 8.31 (m, 1H, NH-C(O)), 6.99-6.84 (4H, C6H4OCH3), 4.76 (m, 1H, CHNHC(O)), 3.86 (s, 3H, C6H4OCH3), 3.24 (m, 2H, SCH2CHNH), 3.16 (bs, 4H, CH2pip), 2.81 (m, 2H, CH2en), 2.74 (bs, 4H, CH2pip), 2.46 (m, 2H, CH2en), 2.61.85 (8H, PCH2CH2N), 2.09 (s, 3H, NCH3), 2.00-1.70 (4s, 12H, PCH3). 31P NMR (CDCl3) δ: 21.3 (broad s), 14.6 (broad s). FT IR (cm-1): [1047 ν (TctN)], [3370 ν (NH)], [2971-2818 ν (CH)], [1652-1636 ν (CO)O, ν NH(CO)], [1240 ν (OCH3)], [747, 726 ν (b, CHdCH)]. Anal. Calcd for C26H46N5O4P2STc: C, 45.55; H, 6.76; N, 10.22; S, 4.68; found: C, 45.3; H, 6.65; N, 10.20; S, 4.65. Syn f Anti Isomerization of [99gTc(N)(2-MPPPcys-OS)(PNP3/4)]. Effect of pH: In a propylene test tube containing EtOH (0.250 mL) and an alcoholic solution of the selected 99gTc-complex (10 mM, 0.050 mL) was added the appropriate buffer solution at pH 5-9 (0.250 mL). The mixture was vortexed and incubated at various temperatures (4 °C, 25 °C, 37 °C) for one week. A control reaction containing an equal volume of water, instead of buffer solution, was maintained in parallel. Aliquots of the reaction mixtures were withdrawn and analyzed by TLC and HPLC chromatography at appropriate time intervals (0 h, 12 h, 24 h, 3 d, 5 d, 7 d). The results are summarized in Table 2. Effect of Temperature: In a propylene test tube containing EtOH (0.250 mL) and an alcoholic solution of the selected 99gTc-complex (10 mM, 0.050 mL), H2O (0.250 mL) was added. The mixture was vortexed and kept at 50 °C or 100 °C for various times (0, 15 min, 60 min, 120 min, 6 and 24 h). Aliquots of the reaction mixtures were withdrawn at the appropriate time and analyzed immediately by TLC and HPLC chromatography (Table 2). Challenge Reaction with Thioacetate: In a propylene test tube containing EtOH (0.250 mL) and an alcoholic solution of 10b (10 mM, 0.050 mL) was added an aliquot of an aqueous solution of sodium O,S-thioacetate (OSTA) (20.0 mM). The volume of the resulting solutions, having final 10b/thioacetate molar ratios of 1/1, 1/5, and 1/10, were adjusted to 0.5 mL with water. The mixtures were vortexed and heated at 50 °C or 100 °C for 24 h. A control reaction containing an equal volume of water without thioacetate was run in parallel. At 15 min, 4 h, and 24 h, aliquots of the reaction mixtures were withdrawn and analyzed by TLC and HPLC chromatography. The results are summarized in Table 3. Preparation of [99mTc(N)(2-MPPP-cys-OS)(PNP3/ 4)] Anti (11a, 12a) and Syn (11b, 12b). Method A: Na[99mTcO4] (0.250 mL, 50.0 MBq-3.0 GBq) was added to a

Table 3. Incubation of syn-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] (10b) with Sodium O,S-Thioacetae (OS-ta) ratio 10b/OS-ta

°C

time

syn-10b

anti-10a

13-(OS-ta)

1/1 1/5 1/5 1/5 1/5

50 50 50 50 100

15 min 15 min 4h 24 h 15 min

93.3 91.4 51.4 4.9 23.3

6.6 7.3 31.6 51.8 68.0

0.1 1.3 17.0 43.3 8.7

vial containing succinic dihydrazide (SDH) (5.0 mg) and SnCl2 (0.1 mg) suspended in saline (0.1 mL) and ethanol (1.0 mL). The vial was kept at room temperature for 15 min giving a mixture of 99mTc-nitrido precursors [99mTct N]mix2+. The appropriate diphosphine ligand (PNP3 or PNP4) (1.0 mg), dissolved in EtOH (0.1 mL), was then added, and the reaction mixture was left standing for 30 min at room temperature. To the resulting intermediate complex [99mTc(N)(PNP3/4)]2+ was added an ethanolic solution (0.1 mL) containing the 2-MPPP-cys-OS ligand (1 mg, 1.6 × 10-3 mmol), and the solution was heated at 100 °C for 30 min. TLC and HPLC characterization showed the presence of two separate radioactive spots, which resulted from the formation of two distinct isomeric forms (a and b) of the complex [99mTc(N)(2-MPPP-cysOS)(PNP3/4)]. Total radiochemical purity (RCP) as determined by TLC and HPLC chromatography for the two isomers was 90.4% for [99mTc(N)(2-MPPP-cys-OS)(PNP3)] (11a, 78.3%; 11b, 12.1%) and 80.7% for [99mTc(N)(2MPPP-cys-OS)(PNP4)] (12a, 65.7%; 12b, 15%). Method B: To a mixture of [TctN]2mix prepared as above, an ethanolic solution (0.1 mL) containing 2-MPPPCys-OS (1 mg, 1.6 × 10-3 mmol) was added, and the reaction vial was left to stand at room temperature for 30 min. The reaction mixture was then treated with PNP3 or PNP4 (1.0 mg) dissolved in EtOH (0.1 mL) and heated at 100 °C for 30 min. Total RCP determined by TLC and HPLC chromatography for the two isomers was 42% for [99mTc(N)(2-MPPP-cys-OS)(PNP3)] (11a, 35.9%; 11b, 6.1%) and 45% for [99mTc(N)(2-MPPP-cys-OS)(PNP4)] (12a, 40.5%; 12b, 4.5%). These products exhibited the same chromatographic profiles as those obtained by method A. Method C: To [TctN]2+mix prepared as above, the appropriate diphosphine (PNP3 or PNP4) (1.0 mg) dissolved in EtOH (0.1 mL) and 2-MPPP-Cys-OS (0.1 mg, 0.16 × 10-3 mmol) dissolved in EtOH (0.1 mL) were added simultaneously, and the reaction mixture was


heated at 100 °C for 30 min. Total RCP determined by TLC and HPLC chromatography was 98% for [99mTc(N)(2-MPPP-cys-OS)(PNP3)] (11a, 85%; 11b, 13%) and 85.4% for [99mTc(N)(2-MPPP-cys-OS)(PNP4)] (12a, 76.9%; 12b, 8.5%). In both cases, the major compound, 11a or 12a, isolated by HPLC (tR 11a ) 23.4 min, tR 12a ) 27.8 min), was concentrated on a Sep pack C18 column rinsed with H2O and eluted using EtOH (3 × 0.5 mL). The second fraction containing all the activity was diluted with saline (1/10 EtOH/saline) and utilized for in vitro stability studies and in vivo biodistribution studies. After this purification the RCP’s of both complexes, evaluated by TLC and HPLC chromatography, was >95%. In all cases the pH of the reaction mixture, measured at the end of the reaction, was 7.5. Preparation of [99mTc(N)(OS-ta)(PNP4)] (OS-ta ) Thioacetate) (13). This complex was synthesized as reported earlier (14). Determination of Partition Coefficients (Log P). After HPLC purification, Log P values of the thermodynamically stable anti-Tc-99m complexes (11a, 12a) were determined by measuring the activity that partitioned between n-octanol (3.0 mL) and aqueous phosphate buffer (3.0 mL, 0.1 M, pH 7.4) under strict equilibrium conditions. Results are presented in Table 1. The syn to anti conversion of 11b and 12b complexes precluded the measurements of their Log P, and these were therefore not determined by this method. Stability Studies. Compound 11a or 12a (100 µL) was added to a 5-mL propylene test tube containing rat serum (900 µL) or saline (900 µL). The resulting mixture was incubated at 37 °C for 2 h and assayed at 15, 30, 60, and 120 min by TLC for any changes in radiochemical purity. The complexes were found to be stable. Glutathione (GSH) and Cysteine Challenge. To a 5-mL propylene test tube containing phosphate buffer (250.0 µL, 0.2 M, pH 7.4) were added water (100.0 µL), 11a or 12a (100.0 µL), and an aliquot (50.0 µL) of an aqueous stock solution of GSH (10.0 mM). The mixture was vortexed and incubated at 37 °C for 2 h. A control reaction containing an equal volume of H2O instead of GSH was studied in parallel. At 15, 30, 60, and 120 min, aliquots of the reaction mixture were withdrawn and analyzed by TLC and HPLC chromatography. The complexes were found to be inert toward substitution by GSH. A study using cysteine hydrochloride (10.0 mM and 1.0 mM) as the challenge ligand demonstrated a similar stability toward cysteine transchelation. Receptor-Binding Assays and Calculations. The 5-HT1A-radioligand-binding assay was performed with rat (Sprague Dawley) cerebral cortex membrane prepared according to published procedures (19, 20). The net content of protein was determined via the Bradford method (Comassie Brilliant Blue, Biorad) using human serum albumin as a standard (21). Competitive binding assays were performed in triplicate using [3H]-8-OH-DPAT as the radioligand, in a final volume of 0.250 mL. Briefly, aliquots (0.1 mL) corresponding to 0.15 mg of membrane protein were mixed with Tris-HCl buffer (50 mM, pH 7.7), [3H]-8-OH-DPAT (0.025 mL, 1 nM final concentration), and increasing concentrations (0.125 mL, 1 nM-10 µM) of the competing 99g Tc-complex (9a, 9b, 10a, and 10b). Incubations were carried out at 25 °C for 30 min. The reaction was terminated by the addition of ice-cold assay buffer (3 mL) followed by separation of bound from free radioligand by rapid filtration of the reaction mixture through Whatman GF/B glass-fiber filters. The filters were rinsed three times with ice-cold Tris-HCl buffer (3 × 3 mL). Filter-

Bolzati et al. Table 4. Inhibition Constant, Ki (nM), for the Displacement of 1.0 nM [3H]-8-OH-DPAT from Rat Cereberal Cortex Membranes 5HT1A Receptors compound

Ki (nM)

2-MPP anti-[99gTc(N)(2-MPPP-cys-OS)(PNP3)] 9a syn-[99gTc(N)(2-MPPP-cys-OS)(PNP3)] 9b anti-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] 10a syn-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] 10b

150 (( 12) >10000 >10000 800 ((17) >10000

bound radioactivity (99gTc) was measured by liquid scintillation counting using a beta counter (LS 5081, Beckman) with a window set to exclude the interference from the tritium channel. IC50 values were obtained graphically and the inhibition constant (Ki) was calculated according to the Cheng and Prussoff equation (22) (Table 4). Animal Studies. Female Sprague-Dawley rats weighing 200-250 g were anesthetized with an intramuscular injection of a mixture of ketamine (80 mg kg-1) and xylazine (19 mg kg-1). Compound 12a (100 µL, 300-370 kBq) was injected via the jugular vein. The animals (n ) 3) were sacrificed by cervical dislocation at 0, 2, 10, 20, and 60 min postinjection. Immediately after sacrifice of the rats, blood was withdrawn from the heart through a syringe and counted. Organs were excised, rinsed with saline, weighed, and counted in a gamma counter. The results are expressed as percentage injected dose per gram (% ID g-1) for each organ and blood (Table 5). RESULTS

Preparation of the Bifunctional Ligand. As outlined in Scheme 1, 2-{3-[4-(2-methoxyphenyl)piperazin1-yl]propionylamino}-3-tritylsulfanylpropionic acid (5) was obtained by coupling the amino group of the S-tritylcysteine ethyl ester (3) to the carboxylic group of 3-[4(2-methoxyphenyl)piperazin-1-yl)]propionate (2) in CH2Cl2 in the presence of EDC, followed by base hydrolysis of the ester group. Compound 2 was obtained by a twostep reaction through N-alkylation of 1-(2-methoxyphenfyl)piperazine hydrochloride (2-MPP) with methyl 3-bromopropionate, and subsequent hydrolysis to the corresponding acid under basic conditions. The final product 2-MPPP-cys-OS (6) was activated by removing the Strityl protecting group from cysteine thiolate with TFA in the presence of triethylsilyl hydride to produce the free thiol ligand. The resulting bifunctional ligand is composed of a potentially bioactive moiety with affinity for 5HT1A receptors tethered to a bidentate chelating system having the thiol and carboxyl oxygen atoms of the cysteine as π-donor coordinating atoms. Preparation and Characterization of 99gTc-labeled Compounds. The intermediate compounds [99gTc(N)Cl2(PNP3/4)] (7, 8) have been prepared, in high yield, via ligand-exchange reaction of the labile precursor [99gTc(N)Cl2(PPh3)2] with the appropriate diphosphine ligand in CH2Cl2 solution (Scheme 2). IR absorption spectra exhibit typical medium intense bands at 1054 (7) and 1045 (8) cm-1, attributable to ν[TctN] along with characteristic absorptions of coordinated alkyldiphosphine ligand in the 2979-2808 cm-1 region. Elemental analyses, as reported in the Experimental Section, are also in good agreement with the proposed formulation. Magnetic equivalence of the diphosphine phosphorus, results in 31P NMR spectra of the intermediate complexes 7 and 8 exhibiting a broad singlet at room-temperature centered at δ ) 31.0 and 23.1, respectively. Similar signal broadening is found in diamagnetic d2 Tc-complexes and is attributed to the quadrupole relaxation induced by the



Table 5. Biodistribution in Rats of 12a (% I.D. dose/g) organ

0 min

2 min

10 min

20 min

30 min

60 min

blood brain heart lungs liver stomach spleen kidneys intestine muscle

3.65 ( 0.25 0.29 ( 0.03 1.83 ( 0.15 1.72 ( 0.09 2.7 ( 0.49 0.65 ( 0.08 0.74 ( 0.03 2.16 ( 0.18 2.00 ( 0.04 0.14 ( 0.04

1.42 ( 0.13 0.14 ( 0.02 0.91 ( 0.15 1.01 ( 0.07 3.22 ( 0.01 0.50 ( 0.04 0.64 ( 0.05 5.77 ( 0.34 6.37 ( 0.04 0.18 ( 0.02

0.53 ( 0.13 0.04 ( 0.01 0.33 ( 0.0 0.4 ( 0.01 2.08 ( 0.02 1.06 ( 0.30 0.20 ( 0.01 1.50 ( 0.21 10.80 ( 0.40 0.10 ( 0.02

0.22 ( 0.02 0.02 ( 0.00 0.16 ( 0.02 0.21 ( 0.02 0.79 ( 0.17 4.25 ( 2.16 0.12 ( 0.00 0.70 ( 0.01 16.65 ( 1.13 0.08 ( 0.01

0.18 ( 0.08 0.02 ( 0.00 0.08 ( 0.00 0.09 ( 0.00 0.40 ( 0.01 2.42 ( 1.39 0.06 ( 0.00 0.50 ( 0.07 13.33 ( 1.51 0.05 ( 0.00

0.01 ( 0.01 0.01 ( 0.00 0.07 ( 0.01 0.08 ( 0.00 0.38 ( 0.03 4.75 ( 1.41 0.06 ( 0.01 0.46 ( 0.05 18.31 ( 2.09 0.08 ( 0.0

Scheme 1

Tc nucleus (I ) 9/2) on the neighboring P atoms (16). H and 13C spectra show relatively complex patterns consistent with the molecular structure depicted in Scheme 2, in which the asymmetry introduced by the terminal nitrido group removes the equivalence of the substituents on P (syn and anti oriented) and makes the methylene protons of the diphosphine chain diastereotopic. The molecular structure of an isostructural Re complex, [Re(N)Cl2(PNP2)] (PNP2 ) bis[(2-diphenylphosphino)ethyl]methoxyethylamine), has recently been solved by X-ray diffraction analysis (15), and the spectroscopic properties exhibited by this Re complex compared well with the complexes depicted in Scheme 2. As detailed in Scheme 2, these intermediate compounds react with the bidentate dinegative ligand 2-MPPP-cys-OS resulting in stable neutral asymmetrical compounds of the type [99gTcN(2-MPPP-cys-OS)(PNP3/ 99 1

4)] (9, 10). The same heterocomplexes are formed with the simultaneous addition of the two polydentate ligands (PNP3/4 and 2-MPPP-cys-OS) to the labile precursor [99gTc(N)Cl2(PPh3)2] in CH2Cl2 solution in the presence of an excess of Et3N. Under the synthetic conditions reported, no symmetric disubstituted compounds of the type [99gTc(N)(PNP3/4)2] and [99gTc(N)(2-MPPP-cys-OS)2] have been detected. For [99gTcN(2-MPPP-cys-OS)(PNP3/4)], two distinct isomeric forms have been isolated and identified as syn or anti isomers depending on the orientation of the N-substituted cysteine pendant group with respect to the central TctN terminal core. The final isomeric ratio, evaluated by TLC and HPLC chromatography, is approximately 50/50 for 9a and 9b and 70/30 for 10a and 10b. In both cases, the two isomers were separated by silica column chromatography and were relatively stable


Bolzati et al.

Scheme 2

on this time scale. IR spectra of these asymmetric Tc(V)-nitrido complexes show typical absorptions of the intermediate compounds with two additional vibrations at 1732-1638 and 1240 cm-1, characteristic of 2-MPPPcys-OS, indicating that the bifunctional chelating ligand is coordinated to the metal fragment [99gTcN(PNP3/4)]2+. The expected bands of the technetium nitrogen multiple bond, appear in the region 1050-1047 cm-1, but no significant difference in the TctN stretch between syn and anti species has been observed. The complete characterization of two isomeric forms was obtained by multinuclear NMR spectroscopy. Coordination of the asymmetric S,O-cysteine fragment on the equatorial plane removes the magnetic equivalence of the facing diphosphine phosphorus. Consequently, the 31P NMR spectrum of each nitrido heterocomplex has two distinct broad signals. Syn and anti isomers are distinguished by the different proton patterns of the coordinated cysteine framework, as previously determined in similar isomeric pairs, [99gTc(O)(RP294)] (RP294 ) dimethylglycyl-L-seryl-L-cysteinylglycinamide) (23) and [Re(N)(PNP2)(N-ac-cys)] (PNP2 ) aryldiphosphine and N-ac-cys ) N-acetyl-L-cysteine) (24), containing N,Scarboxylic-substituted cysteine and O,S-amine-substituted cysteine, respectively. The assignment of the anti isomer in complexes 9 and 10 is based on the assumption that the pertinent NH and methyne protons of cysteine are shifted upfield and downfield, respectively, relative to the corresponding proton resonances in the syn isomer (Figure 2).

The syn f anti isomerization of 9b and 10b was found to occur relatively slowly and to be an irreversible and temperature-dependent process. In fact, by monitoring the conversion by TLC or HPLC for 10b (see Table 2), we observed that the isomerization reached the final 90/ 10 anti/syn ratio only after one week at 37 °C, and after only 15 min at 100 °C. A faster syn f anti conversion was observed for the syn-PNP3 complex. On the other hand, no variation of the reverse anti f syn conversion was observed by either varying the pH of the mixture from 5 to 9 or the temperature.

Figure 2. Proton NMR spectra in the 8.4-3.5 ppm region of anti-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] 10a (top), of syn-[99gTc(N)(2-MPPP-cys-OS)(PNP4)] 10b (bottom), and mixture of 10a and 10b (middle).



Scheme 3

To clarify the mechanism of isomer conversion, syn[99gTcN(2-MPPP-cys-OS)(PNP4)] (10b) was incubated with an equimolar or excess amount of the less hindered cysteine-mimicking molecule O,S-thioacetate sodium salt at 50 °C and 100 °C. As reported in Table 3, in addition to the expected syn f anti conversion, the chromatographic profile reveals the presence of a more hydrophilic peak (see Scheme 4) identified as [99gTcN(O,S-thioacetate)(PNP4)] (13) by comparison with an authentic sample prepared in earlier studies (17). The production of 13 is increased with time by using an excess of O,Sthioacetate or by raising the temperature. Preparation of 99mTc-Labeled Compounds. Methods employed for preparing neutral tracer asymmetric 99m Tc-nitrido complexes of the type [99mTc(N)(PNP3/4)(2-MPPP-cys-OS)] are outlined in Scheme 3. The labeling procedures have been carried out using either a two- or a three-step approach. In all preparations the first step is to generate a mixture of 99mTc-nitrido precursors, all containing the [TctN]2+ core, through the reduction of pertechnetate with tin(II) chloride in the presence of SDH as donor of the nitrido nitrogen atom. The three-step procedure (method A) entails formation of the strongly electrophilic [99mTc(N)(PNP)]2+ intermediate fragment by addition of the appropriate diphosphine ligand (PNP3/4) to the starting 99mTc-nitrido mixture. The preparation is then completed by addition of 2-MPPP-cys-OS, which selectively reacts with the intermediate fragment to yield the

final asymmetric complexes. An analogous three-step procedure (method B) reverses the order of addition of the reagents. The yield, however, is somewhat reduced. The optimal labeling procedure involves only two separate steps (method C). After production of the mixture [99mTctN]mix2+, the appropriate diphosphine and bidentate 2-MPPP-cys-OS ligand are simultaneously added to the reaction vial to afford the final product in high yield, after 30 min at 100 °C. Chromatographic characterization indicates that the chemical nature of the final complexes is independent of the preparation method utilized. The chemical identity of the 99mTc-complexes were established by comparison of their chromatographic properties with those of the corresponding complexes obtained at the macroscopic level with the long-lived 99gTc isotope (see Table 1 and Figure 3). The log k0′ values were determined for each compound by reversed-phase HPLC using various mixtures of methanol/buffer (pH 7.4) as eluant, these served as a measure of the relative lipophilicity of the complexes particularly when traditional octanol/water (Log P) ratios (Table 1) could not be determined due to syn fanti isomer conversion. All 99mTc-complexes exist in two distinct isomeric forms (see 99gTc) but with an anti/syn isomeric ratio of 90/10 irrespective of the diphosphine utilized. This difference in the tracer 99mTc synthesis, compared with the macroscopic 99gTc synthesis is attributed to the higher temperature employed in the nca 99mTc synthesis which accel-

Figure 3. RP-HPLC comparison of [99mTc(N)(2-MPPP-cys-OS)(PNP4)] (12) complexes with [99gTc(N)(2-MPPP-cys-OS)(PNP4)] (10) analogue.


erates the syn to anti conversion observed at room temperature with the 99gTc-complexes. Consequently, only the anti 99mTc isomers were isolated by HPLC (RCP > 95%) and utilized for both in vitro stability and in vivo biodistribution studies after appropriate dilution with saline. Stability. Complexes 11a and 12a exhibit a good in vitro stability and no significant change in radiochemical purity after 2 h of incubation at 37 °C, thus indicating the high stability and substitution inertness of these compounds. Biological Studies. The in vitro affinity for the 5HT1A receptors of the underivatized 2-MPP compound and of the technetium complexes (9a, 9b, 10a, 10b) was assessed by measuring the ability of the compounds to compete with [3H]-8-OH-DPAT binding in isolated membranes from rat cerebral cortex. The inhibition bindingconstant (Ki) values, measured at 25 °C (Table 4), show that only complex 10a (anti-PNP4) possesses some affinity compared with that observed for the free 2-MPP ligand, while a complete absence of receptor affinity is observed for the other complexes. For biodistribution studies of the 99mTc-compound 12a in female Sprague-Dawley rats, the radiolabeled compound was purified before injection to remove excess unlabeled cold ligand. The radiochemical purity of the purified compound was verified prior to in vivo administration and the tissue distribution of the complexes is summarized as % ID/g tissue in Table 5. The complex exhibits rapid blood clearance with the activity mainly eliminating through both the hepatobiliary system and the urinary tract. Only a small fraction of the injected activity (=0.14% ID at 2 min) crosses the BBB, followed by a rapid wash out. A very high intestinal uptake is observed at 2 min postinjection. DISCUSSION

In this study a new route for incorporating a bioactive molecule into stable 99mTc-nitrido compounds is reported. The method is based on the chemical properties of the electrophilic nitrido metal fragment [99g/99mTcN(PNP)]2+. The structural framework of these moieties contains a pseudo tridentate diphosphine ligand (PNP) coordinated to the technetium-nitrido group and two labile sites usually filled by halogens which complete the pseudooctahedral environment. The [99g/99mTcN(PNP)]2+ metal fragment is an activated intermediate which selectively reacts with bidentate chelating ligands carrying π-donor atoms (YZ) to form the final complex [Tc(N)(YZ)(PXP)]0/+ which is characterized by an asymmetric arrangement of two polydentate ligands around the metal center. Neutral asymmetric complexes [99gTcN(2-MPPP-cysOS)(PNP)] are prepared in high yield by ligand-exchange reactions of [99gTc(N)Cl2(PNP)] with the bifunctional ligand 2-MPP-cy-OS, or, alternatively, by direct reaction of the relevant diphosphine (PNP3 or PNP4) and 2-MPPPcys-OS with the labile precursor [99gTc(N)Cl2(PPh3)2]. As indicated in Scheme 2, the heterocomplexes display a metal fragment, composed of a terminal TctN multiple bond with an ancillary diphosphine ligand, and are coordinated to the thiolate sulfur and carboxylate oxygen of the 2-MPPP-cys-OS ligand. Upon coordination to the metal fragment, 2-MPPP-cys-OS adopts two distinct isomeric forms depending on the relative syn or anti orientation of the O,S-cys-piperazinyl group with respect to the TctN terminal group. The isolated syn isomer slowly and irreversibly converts to the anti isomer in a temperature-dependent process.

Bolzati et al.

pH does not modify the course of this syn f anti conversion (Table 2). This lack of acid-base sensitivity is likely due to the pseudo-octahedral geometry of the nitrido heterocomplex, where the position trans to the nitrido group is already occupied by the PNP tertiary amine nitrogen, thus preventing the insertion of additional molecules (e.g., water). A dissociative mechanism for this syn f anti conversion is proposed based on the temperature dependence of the conversion process and the formation, in addition to syn f anti transformed complex, of a more hydrophilic [Tc(N)(OS-ta)(PNP)] species when the syn complex is incubated with equimolar or excess amounts of a less hindered cysteine-mimicking S,O-thioacetate (Scheme 4). These observations support a mechanism which requires the dissociation of the carboxylate oxygen of 2-MPPP-cys-OS and the insertion of the thioacetate thiolate, resulting in production of the hydrophilic [Tc(N)(OS-ta)(PNP)] species together with isomerization of the syn to anti [Tc(N)(2-MPPP-cys-OS)(PNP)]. This behavior also suggests that the anti isomer is the sterically and thermodynamically favored conformation of these heterocomplexes. The chromatographic profile (Figure 3) shows that the complexes synthesized at the millimolar level are identical to those prepared at n.c.a. level, the only difference being the increased anti/syn isomeric ratio, which is attributed to the higher temperature employed in the former 99mTc synthesis. At the n.c.a. level asymmetric 99m Tc-nitrido complexes are efficiently prepared through three alternative routes as indicated in Scheme 3. The most efficient labeling procedure involves only two steps, mixing in the same reaction vial both the PNP and 2-MPPP-cys-OS ligand with the starting mixture of [99mTctN]2+mix nitrido precursor. Alternatively, intermediate Tc-nitrido diphosphine complex can also be formed prior to reacting with the π-donor 2-MPPP-cys-OS ligand present in the same reaction vial to give the desired nitrido heterocomplex in high yield. The most salient feature of these reactions is that no formation of the symmetric complexes [99mTc(N)(PNP)2]2+ and [99m Tc(N)(2-MPPP-cys-OS)2] are detected under a broad range of experimental conditions. In vitro stability studies carried out on the anti isomers show that these compounds are stable in aqueous solutions and serum and are inert toward transchelation with free glutathione and cysteine, suggesting that the arrangement of two π-acceptor phosphorus and two π-donor atoms around the [TctN]2+ core possess a high thermodynamic stability and kinetic inertness. Despite these promising physicochemical properties, in vitro binding studies of the technetium complexes (9a, 9b, 10a, 10b) with the 5HT1A receptors show that only complex 10a (anti-PNP4) retains some affinity for the 5HT1A receptors. The complete loss of receptor affinity observed for complexes containing the PNP3 phosphine could be attributed to the larger steric hindrance imparted to the metal fragment by this phosphine. The relatively smaller complex formed with PNP4 displayed Ki values of the same order of magnitude as the underivatized 2-MPP ligand (22). For PNP4 complexes, the partial loss of affinity may be attributed to the interaction of the piperazinyl fragment with the aminomethyl PNP pendant group. This suggests that affinity could be improved by further manipulating the structure, e.g., by increasing the length of the spacer between the piperazine moiety and the cysteine fragment, eliminating the amide link in the linker arm, or using an alkyl link between the MPP and the bidentate coordinating groups. Studies with this focus are in progress.



Scheme 4

Although both the molecular weight and the lipophilicity of the anti-PNP4 complex are in the appropriate range required for crossing the BBB, in vivo biodistribution data displayed negligible brain uptake. This low affinity toward the receptor along with the rapid blood clearance of the complex may reduce the bioavailability of the tracer thus limiting the brain uptake. In non-CNS peripheral sites, such as the intestine, in our opinion, the accumulation may reflect an excretory pathway via the hepatobillary system for these molecule rather than binding to peripheral 5HT1A receptors, particularly since the complex displays only low affinity toward the receptor in vitro.

experimental evidence that the incorporation of the [Tct N(PNP4)]2+ molecular fragment with a bioactive molecule (2-MPP) maintains its affinity for 5HT1A receptors suggests that this class of complexes might be useful for peripheral applications (25) and that this strategy may be applied to other receptor-targeting molecules with appropriately modified bidentate chelates. ACKNOWLEDGMENT

The authors are indebted to Nihon Medi-Physic, Tokyo, Japan, for financial support of this work. The authors wish to thank Anna Rosa Moresco for her work on the elemental analysis.

CONCLUSIONS

This work describes application of a new labeling procedure for incorporating a bioactive molecule into a stable asymmetric 99mTc-nitrido complex. The chemistry is based on the use of the [TcN(PNP)]2+ metal fragment which, once formed, selectively reacts with bidentate ligands, such as bifunctional N-derivatized cysteine ligand carrying the bioactive 2-MPP fragment. The resulting neutral species are easily prepared both at macroscopic and n.c.a. levels and exhibit remarkable stability toward glutathione or cysteine challenge. Despite these promising properties, the lack of BBB penetration indicates that these particular complexes may not be useful for CNS-receptor mapping. However, the

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BC034100G

The [99mTc(N)(PNP)]2+ Metal Fragment: A Technetium-Nitrido

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