Asymmetrical Nitrido Tc-99m Heterocomplexes as Potential Imaging

Bioconjugate Chem. 2003, 14, 1279−1288

1279

Asymmetrical Nitrido Tc-99m Heterocomplexes as Potential Imaging Agents for Benzodiazepine Receptors Alessandra Boschi,† Licia Uccelli,† Adriano Duatti,*,† Cristina Bolzati,‡ Fiorenzo Refosco,‡ Francesco Tisato,‡ Romeo Romagnoli,§ Pier Giovanni Baraldi,§ Katia Varani,§ and Pier Andrea Borea§ Laboratory of Nuclear Medicine, Department of Clinical & Experimental Medicine, University of Ferrara, 44100 Ferrara, Italy, Department of Pharmaceutical Sciences, University of Ferrara, 44100 Ferrara, Italy, and Istituto di Chimica Inorganica e delle Superfici - CNR, 35127 Padua, Italy. Received July 17, 2003; Revised Manuscript Received August 21, 2003

The design, synthesis, and biological evaluation of nitrido technetium-99m complexes for imaging benzodiazepine receptors are described. The design was performed by selecting the precursor biologically active substrate desmethyldiazepam, and the reactive metal-containing fragment [99mTc(N)(PXP)]2+ (PXP ) diphosphine ligand) as molecular building-blocks for assembling the structure of the final radiopharmaceuticals through the application of the so-called ‘bifunctional’ and ‘integrated’ approaches. This required the synthesis of the ligands H2BZ1, H2C1, and H2C2 (Figures 1 and 2) derived from desmethyldiazepam. In turn, these ligands were reacted with [99mTc(N)(PXP)]2+ to afford the complexes [99mTc(N)(PXP)(L)] (L ) BZ1, C1, C2). The chemical nature of the resulting Tc-99m radiopharmaceuticals was investigated using chromatographic methods, and by comparison with the analogous complexes prepared with the long-lived isotope Tc-99g and characterized by spectroscopic and analytical methods. Results showed that the complexes [99mTc(N)(PXP)(L)] are neutral and possess an asymmetrical five-coordinated structure in which two different bidentate ligands, PXP and L, are coordinated to the same TctN core. With the ligand H2BZ1, two isomers were obtained depending on the syn or anti orientation of the pendant benzodiazepine group relative to the TctN multiple bond. Biodistribution studies of Tc-99m complexes were carried out in rats, and affinity for benzodiazepine receptors was assessed through in vitro binding experiments on isolated rat’s cerebral membranes using the corresponding Tc-99g complexes.

INTRODUCTION

Benzodiazepine receptors are ubiquitously involved in the GABA postsynaptic receptor complex. In particular, the biochemical evidence indicates that benzodiazepine receptors are part of the GABAA receptor chloride ion channel complex. Alterations of benzodiazepine receptor distribution are involved in many pathological states of the central nervous system (1-4). Positron emission tomography (PET) and single photon emission tomography (SPET) are unique technologies for the noninvasive monitoring of receptor patterns in integrated organisms without affecting their in vivo stability. Various radiopharmaceuticals have been proposed as suitable candidates for the application of these imaging procedures to the evaluation of benzodiazepine receptor distribution (5, 6). It has been showed previously that the derivative Iomazenil labeled with the γ-emitting radionuclide I-123 possesses useful biological characteristics, and it is currently employed as imaging agent for the diagnosis of various brain diseases related to alterations of benzodiazepine receptors (7). Nonetheless, since production of I-123 is performed through a neutron reactor, its routine clinical use is strongly limited. Analogous compounds labeled with the short-lived, positron-emitting nuclides C-11 and F-18 have been also described (8-13). However, * Author for correspondence. Phone: 39-0532-236545. Fax: 39-0532-236589. E-mail: [email protected]. † Department of Clinical & Experimental Medicine. ‡ ICIS, CNR. § Department of Pharmaceutical Sciences.

the main limitation with this type of radionuclides comes from the fact that their production requires the use of an on-site cyclotron, an apparatus that is not widely accessible in all nuclear medicine centers. The γ-emitting nuclide technetium-99m possesses almost ideal nuclear properties for SPET imaging and is readily available in all nuclear medicine centers through the use of a transportable 99Mo/999mTc generator (14). These advantages account for the widespread use of Tc99m radiopharmaceuticals in routine clinical practice. They further suggest that the search for a useful Tc-99m tracer for imaging benzodiazepine receptor distribution would be highly desirable. However, the preparation of a diagnostic agent of this type would appear immediately as a rather difficult task due to the strict, structural requirements that should be attained by such a compound in order to penetrate the intact blood-brain barrier (BBB) and localize selectively into the target receptor. For instance, a first prerequisite indicates that diffusion through BBB can be achieved only for complexes having a neutral charge and a suitable lipophilic character. Moreover, the molecular shape of the potential Tc-99m receptor imaging agent has to fit correctly the target receptor site to allow the setting up of a selective interaction (15-17). The above considerations clearly point out the need for a preliminary design of the structure of a Tc-99m complex before coming to its actual synthesis. In the last years, two main approaches to the design of Tc-99m receptor imaging agents have been proposed. Both strategies start initially with the selection of a convenient biologically

10.1021/bc034124n CCC: $25.00 © 2003 American Chemical Society Published on Web 09/27/2003

1280 Bioconjugate Chem., Vol. 14, No. 6, 2003

active molecule or drug know to have affinity for a specific receptor. After this common input, the two methods diverge. The so-called ‘bifunctional’ or ‘pendant’ approach literally suggests to append the bioactive group to a metal complex through a suitable linkage. This can be conveniently accomplished using a ‘bifunctional ligand’ that could be viewed as a composite molecule combining a strong chelating group for the radiometal with the biomolecule by means of a suitable linker connecting these two moieties. After the radiometal has been encaged by the chelating system, the resulting ‘conjugate’ complex retains the bioactive group into its structure as an appended side chain. Instead, the other procedure, called ‘integrated approach’, puts its focus on the selected biomolecule itself that may serve as a mold for assembling the structure of the final Tc-99m receptor radiopharmaceutical. The key step is to identify a region in its structure that is not essential for preserving its biological properties. The final radiopharmaceutical, therefore, is assembled by replacing this nonessential part with a metal-containing fragment having a molecular shape and dimension similar to the substituted portion of the original biomolecule in order to fit almost exactly into the same position. Obviously, the ultimate success of both design strategies lies in their ability to keep unaltered the intrinsic biological behavior of the starting biomolecule (17, 14, 18-20). A key, theoretical advantage of the two approaches outlined above originates from their representation of the molecular structure of a radioactive tracer as consisting of different ‘pieces’ or ‘fragments’, which can be, at least in principle, conveniently assembled to build up the final radiopharmaceutical. However, the merging of the various fragments to yield a stable product is not always simple to accomplish and, for practical purposes, this has been obtained only through the application of the bifunctional approach. In recent years, an alternative approach to the problem of assembling the various parts of a receptor-specific Tc-99m radiopharmaceutical has emerged. This method is based on the chemical properties of certain types of substitution-labile technetium complexes showing a marked reactivity only toward ligands having some specific set of coordinating atoms. In these complexes, a few coordination positions are occupied by a set of ligands that are tightly bound to the metal center. The resulting strong ligand field allows a significant stabilization of the metal oxidation state preventing the complex to undergo oxidation-reduction reactions. The remaining positions of the coordination arrangement are usually spanned by weakly bound ligands that could be easily replaced by some other incoming ligand carrying a specific set of donor atoms. As a consequence, the reaction between the precursor complex and the hypothetical incoming ligand is expected to be kinetically favored and should produce the final substituted complex in very high yield. Such a behavior can be efficiently exploited for assembling a ‘robust’ Tc-99m fragment with a biomolecule including the appropriate set of coordinating atoms. The high affinity of the precursor metal fragment for the specific donor set on the bioactive ligand would ensure the perfect fitting of these two molecular building blocks to form the final, combined complex (21). The first example of the application of the above ‘metal fragment’ approach have been reported, a few years ago, by Alberto and co-workers. This is based on the chemical properties of the precursor, aquo-carbonyl, metal complex [99mTc(CO)3(H2O)3]+. In this species, the fragment [99mTc(CO)3]+ constitutes the chemically inert portion of

Boschi et al.

Figure 1. Chemical structure of complexes 1-3.

the complex. On the contrary, the three water molecules are weakly coordinated and, thus, can be easily replaced by some substituting ligand having the appropriate set of donor atoms. Under these conditions, the reaction between the metal fragment [99mTc(CO)3]+ and the incoming ligand becomes highly selective allowing the efficient assemblage of these two pieces to afford the final complex. Therefore, the precursor complex [99mTc(CO)3(H2O)3]+ can be conveniently used as a synthon for the synthesis of a variety of Tc-99m carbonyl derivatives (22-24). Very recently, we described the second definite example of the ‘metal fragment’ approach by studying the synthesis of a novel class of asymmetrical nitrido heterocomplexes characterized by the presence of two different bidentate ligands bound to the same Tc5+ center. It was found that this novel type of mixed-ligand complexes was efficiently prepared by reacting the precursor complex [99mTc(N)(PXP)Cl2] (PXP ) diphosphine ligand) with bidentate chelating ligands carrying π-donors as coordinating atoms. In these reactions, the arrangement of atoms [99mTc(N)(PXP)]2+, composed by a TctN group coordinated to a chelating diphosphine ligand PXP, behaves as ‘robust’ metal fragment, and the two chlorine atoms can be easily displaced by the incoming π-donor ligand to afford the asymmetrical complex [99mTc(N)(PXP)(L)]0/+. Thus, the metal synthon [99mTc(N)(PXP)]2+ could be conveniently utilized to obtain a very broad class of asymmetrical nitrido Tc(V) complexes with a variety of bidentate ligands (25-27). In this paper, we describe the application of the labeling method based on the metal fragment [99mTc(N)(PXP)]2+ to the preparation of a series of Tc-99m complexes with bidentate ligands derived from the benzodiazepine receptor drug desmethyldiazepam showed in Figure 1. This latter compound has been modified in two different ways to allow the application of both the bifunctional and integrated approaches as described in the following sections. The resulting ligands H2BZ1, H2C1, and H2C2 were then reacted with [99mTc(N)(PXP)]2+ to afford asymmetrical Tc-99m complexes, which have been characterized by chromatographic methods and their biological properties evaluated both in vitro and in vivo. A drawing of the structure of the new complexes and the corresponding numbering scheme are illustrated in Figures 1 and 2 for the ligands H2BZ1, and H2C1 and H2C2, respectively. EXPERIMENTAL SECTION

All reactions were carried out under an inert atmosphere of dry nitrogen, unless otherwise described. Organic solutions were dried over anhydrous Na2SO4. Dry DMF was distilled from calcium chloride and stored over molecular sieves (3 Å).

Tc-99m Imaging Agents for Benzodiazepine Receptors

Bioconjugate Chem., Vol. 14, No. 6, 2003 1281 Scheme 1. Schematic Drawing of the Preparation of the Ligand H2BZ1a

a Reagents and conditions: (a) NaH, BrCH CO Et; (b) LiOH, 2 2 THF/MeOH/H2O (3:1:1); (c) EDCl, HOBt, S-trityl cysteine tertbutyl ester, DMF, rt, 18 h; (d) Et3SiH, TFA, DCM, rt.

Figure 2. Chemical structure of the ligands H2C1 and H2C2, and of complexes 3 and 5.

Figure 3. The PNP-type diphosphine ligands.

The diphosphine ligands PNP2, PNP3, and PNP4, shown in Figure 3, were prepared as reported elsewhere or obtained from Argus Chemicals (Florence, Italy). The ligand H2BZ1 was obtained using a procedure detailed below and illustrated in Scheme 1. The ligands (L)-Nthiobenzoyl glicine (H2C1) (28) and (L)-N-thiobenzoyl leucine (H2C2) (29) (Figure 2) have been synthesized following literature procedures. Succinic dihydrazide [SDH ) H2NNH(OC)CH2CH2(CO)NHNH2] and SnCl2‚ 2H2O were purchased from Aldrich Chimica (Milan, Italy). A generous supply of the compound 7-chloro-1,3-

dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one (desmethyldiazepam or nordazepam) was obtained as a gift from FIS S.p.A. (Vicenza, Italy). [3H]-Ro151788 (85.0 Ci/mmol) and [3H]-flunitrazepam (85.0 Ci/mmol) were provided by New England Nuclear. Tc-99g as [NH4][99gTcO4] was obtained from Oak Ridge National Laboratory. Samples were dissolved in water and treated with excess aqueous ammonia and 30% hydrogen peroxide at 80 °C prior to use to eliminate residual TcO2. Solid samples of purified ammonium pertechnetate were obtained by slow evaporation of the solvent by heating at 40 °C. Caution! Tc-99g is a weak β-emitter (Eβmax ) 0.292 MeV, t1/2 ) 2.12 × 105 years). When handled in milligram amounts, it does not present a serious health hazard as normal laboratory glassware provides adequate shielding. Bremmstrahlung is not a significant problem due to the low energy of the β-particle. However, a laboratory approved for low-level radiation equipped with gloves-boxes and monitored hoods, and normal radiation safety procedures should be used to prevent contamination and inhalation. The complex [99gTc(N)Cl2(PPh3)2] was prepared as described previously (30). TLC chromatography was performed on Merck silica gel plates. TLC chromatograms were analyzed with a Packard Cyclone instrument equipped with a phosphor imaging screen and OptiQuant image analysis software. HPLC was performed on a Beckman System Gold Instrument equipped with a programmable solvent Module 126, a scanning detector Module 166, and a radioisotope detector Module 170. Chromatographic runs were performed on a reversed-phase C18 column (Beckman Ultrasphere, 4.6 × 250 mm) with a reversed- phase C18 precolumn (Beckman Ultrasphere, 4.6 × 45 mm) or, alternatively, on a semipreparative reversed-phase C18 column (Beckman Ultrasphere, 10.0 × 250 mm). The radiochemical purity (RCP) of Tc-99m complexes was expressed as the ratio between the activity associated with a single radiocompound, revealed as a single spot on a TLC strip, and the total activity on the strip or, alternatively, as the percent of the area under the corresponding HPLC peak. Positive ion fast atom bombardment mass spectra (FAB+) of selected complexes in an NBA matrix were


recorded on a VG 30-250 spectrometer (VG Instrument) at the probe temperature. Xe was used as the primary beam gas and the ion gun was operated at 8 keV (ca. 1.28 × 10-15 J) and 100 µA. Elemental analyses (C, H, N, S) were carried out 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 in KBr mixtures using a Spectra-Tech diffusereflectance collector accessory. IR spectra of the ligands H2BZ1, H2C1, and H2C2 were recorded on a Perkin-Elmer 781 IR spectrophotometer. Proton and 31P NMR spectra were collected in CDCl3 and d6-DMSO solutions on a Bruker AC-200 instrument using SiMe4 as internal reference (1H) and 85% aqueous H3PO4 as external reference (31P). Preparation of (7-Chloro-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]diazepin-1-yl)acetic Acid Ethyl Ester (II). A mixture of sodium hydride (50% dispersion in mineral oil, 288 mg, 6 mmol) and desmethyldiazepam (I) (1.35 g, 5 mmol) in DMF (5 mL) was stirred at room temperature for 0.5 h. Ethyl bromoacetate (1 g., 6 mmol) in DMF (2 mL) was then added, and the reaction mixture was stirred further for 18 h at room temperature and decomposed with ice. The resulting mixture was extracted with EtOAc (2 × 15 mL) and the organic phase washed with water (10 mL), brine (5 mL) and finally dried over Na2SO4. After evaporation under vacuum, the residue was purified by flash-chromatography (EtOAc/ petroleum ether, 7:3, v/v) to give II (1.48 g, yield ) 81%) as a white solid. Preparation of (7-Chloro-2-oxo-5-phenyl-2,3-dihydro-benzo[e][1,4]diazepin-1-yl)acetic Acid (III). Lithium hydroxide monohydrate (252 mg, 6 mmol) was added to a solution of II (732 mg, 2 mmol) in 15 mL of THF/MeOH/H2O (3:1:1) at room temperature. The reaction mixture was stirred for 3 h, concentrated under vacuum, and then diluted with water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with brine (5 mL), dried over Na2SO4, and concentrated under vacuum. Flash chromatography (EtOAc) afforded III (494 mg, yield ) 75%) as a white solid. Preparation of (S)-2-[2-(7-Chloro-2-oxo-5-phenyl2,3-dihydro-benzo[e][1,4]diazepin-1-yl)acetylamino]3-tritylsulfanylpropionic Acid tert-Butyl Ester (IV). EDC (960 mg, 5 mmol, 1.25 equiv), HOBt (675 mg, 5 mmol), and S-trityl cysteine tert-butyl ester (1.68 g., 4 mmol) were added to a solution of III (1.31 g, 4 mmol) in dry DMF (10 mL) cooled at 0 °C (29). This mixture was stirred for 24 h and then concentrated in vacuo. The residue was dissolved in EtOAc (10 mL) and washed with water (5 mL) and brine (5 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The resulting residue, purified by column chromatography using EtOAc/CH2Cl2 (2:8, v/v), furnished the derivatives IV (1.82 g, yield ) 62%) as a white solid. Preparation of (S)-2-[2-(7-Chloro-2-oxo-5-phenyl2,3-dihydro-benzo[e][1,4]diazepin-1-yl)acetylamino]3-mercaptopropionic Acid (V). The compound IV (730 mg, 1 mmol) was stirred for 3 h, at room temperature, in a mixture of TFA/CH2Cl2 (1:1, 5 mL) containing Et3SiH (0.8 mL, 582 mg, 5 equiv). The volatiles were removed in vacuo, and the residue was diluted with 5% aqueous NaHCO3 (5 mL). The aqueous mixture was extracted with CH2Cl2 (3 × 5 mL), and the combined organic extracts were dried over Na2SO4 and concentrated in vacuo. The resulting pale-yellow solid was used for the next reactions without any further purification.

Boschi et al.

(V) C20H18N3O4SCl. Calcd (%): C, 55.62; H, 4.20; N, 9.73; S, 7.41. Found (%): 55.68; H, 4.25; N, 9.69; S, 7.20. Synthesis of the Intermediate Complexes [99gTc(N)(PXP)Cl2] (PXP ) PNP2, PNP3, PNP4). The dichloride derivatives of the metal synthon [99gTc(N)(PXP)]2+ were obtained starting from the precursor nitrido complex [99gTc(N)Cl2(PPh3)2]. A representative procedure is given below for the ligand PNP3. [99gTc(N)Cl2(PPh3)2] (94 mg, 0.132 mmol) was suspended in dicloromethane (10 mL) and then reacted with PNP3 (73 mg, 0.145 mmol) at reflux temperature, for 30 min. The starting pink-orange solution turned to yellow. The solvent was removed by passing a weak stream of nitrogen, and a yellow solid was collected, which was washed with isopropyl alcohol, diethyl ether, and nhexane. The yellow compound was dissolved in the minimum amount of chloroform, and the resulting solution was treated with diethyl ether. The precipitation of yellow microcrystalline powder was observed, which was then dried by overnight high-vacuum suction. Yields expressed as (number of moles of the final complex/number of moles of the starting complex) × 100 were in the range 75-80%. Synthesis of the Complexes [99gTc(N)(L)(PXP)] (H2L ) H2BZ1, H2C1, H2C2; PXP ) PNP2, PNP3, PNP4) (1-5). The asymmetrical heterocomplexes [99gTc(N)(PXP)(L)] were prepared by reacting the ligand H2L with the intermediate complexes [99gTc(N)(PXP)Cl2]. A representative procedure is given below for the ligands PNP3 and H2BZ1. [99gTc(N)(PNP3)Cl2] (51 mg, 0.077 mmol) was dissolved in 10 mL of a ethanol/dicloromethane mixture (1:1 v/v) under a nitrogen atmosphere. Triethylamine (0.2 mL) was added to the mixture followed by H2BZ1 (40 mg, 0.092 mmol). The reaction solution was refluxed for 60 min while keeping the nitrogen flux. After cooling, the solvent was removed by passing a weak nitrogen stream. A solid was collected and then washed with isopropyl alcohol, diethyl ether, and n-hexane. The resulting compound was dissolved in the minimum amount of acetonitrile, and the resulting solution was filtered and treated with diethyl ether. The slow precipitation of microcrystalline powder was observed, which was washed with diethyl ether and further dried by overnight highvacuum suction. Yields expressed as (number of moles of the final complex/number of moles of the starting complex) × 100 were in the range 70-80%. HPLC chromatography of the yellow products isolated from reactions with the ligands H2BZ1 and PXP (PXP ) PNP2, PNP3, PNP4) revealed that these species were actually composed by a mixture of two different compounds A and B, which were assigned to the syn and anti isomers of the complexes 1-3 (see Results and Discussion). However, the relative yields of the two isomers were found to be dependent on the type of diphosphine ligand utilized. Specifically, when PXP ) PNP2 and PNP3, complex A was obtained almost quantitatively with a relative yield > 95%. On the contrary, with the ligand PNP4, the relative yields of compounds A and B were 79% and 21%, respectively. Spectroscopic data and elemental analyses for Tc-99g complexes are reported in Table 3. Due to the low yields of isomer B for complexes 1-3, only isomer A was fully characterized. Preparation of the Complexes [99mTc(N)(PXP)(BZ1)] (PXP ) PNP2, PNP3, PNP4). Na[99mTcO4] (50 MBq) was added to a vial containing 5.0 mg of SDH, 0.1 mg of SnCl2 (suspended in 0.1 mL of saline) and 1.0 mL


Bioconjugate Chem., Vol. 14, No. 6, 2003 1283

Table 1. IR and 1H NMR Spectral Data of Compounds II-V compd

IR, (KBr) cm-1

II

1737, 1684, 1610, 1486, 1403, 1351, 1324, 1206, 1029, 698 3417, 1670, 1606, 1484, 1414, 1395, 1318, 826 3400, 1680, 1485, 1325, 1153, 698 3421, 1677, 1560, 1484, 1326, 1194, 828

III IV V a

1H

NMR

1.28 (t, J ) 7.2 Hz, 3H), 3.96 (d, J ) 11.2 Hz, 1H), 4.24 (m, 2H), 4.53 (d, J ) 17.2 Hz, 1H), 4.62 (d, J ) 17.2 Hz, 1H), 4.94 (d, J ) 11.2 Hz, 1H), 7.32 (m, 2H), 7.51 (m, 4H), 7.67 (m, 2H)a 3.76 (d, J ) 10.6 Hz, 1H), 4.01 (d, J ) 16.2 Hz, 1H), 4.22 (d, J ) 16.2 Hz, 1H), 4.53 (d, J ) 10.6 Hz, 1H), 7.11 (m, 1H), 7.55 (m, 7H), 13.1 (bs, 1H)b 1.28 (s, 9H), 2.24 (m, 1H), 3.29 (s, 2H), 3.84 (d, J ) 10.6 Hz, 1H), 4.01 (m, 1H), 4.48 (d, 7.8 Hz, 1H), 4.53 (d, J ) 10.6 Hz, 1H), 7.33 (m, 23H), 8.62 (d, J ) 7.2 Hz, 1H)b 2.39 (m, 1H), 2.82 (m, 2H), 3.84 (s, 2H), 4.24 (m, 1H), 4.57 (s, 2H), 7.19 (m, 1H), 7.54 (m, 7H), 8.23 (t, J ) 5.8 Hz, 1H), 13.04 (bs, 1H)b

CDCl3 solutions. b d6-DMSO solutions.

Table 2. Chromatographic and Lipophilicity Data for the Prepared Complexes complex

TLC (Rf)a

HPLC (tR, min)

P

log P

1A, 1B 2A, 2B 3A, 3B 4 5

0.69, 0.57b 0.62, 0.51b 0.41, 0.30c 0.60d 0.50d

43.9, 40.0e 28.4, 25.8f 30.1, 23.1g 28.3g 33.6g

5758, 3167 441, 175 99, 43

3.76, 3.50 2.64, 2.24 1.99, 1.63

a SiO . b EtOH/CHCl /C H (0.7:3:1.5). c EtOH/CHCl3/C6H6 2 3 6 6 (1.5:2:1.5). d EtOH/CHCl3/toluene/NH4Ac (0.5 mol dm-3) (6:3:3:1). e Et N (0.1 mol dm-3) in aqueous H PO (1.0 mol dm-3) (45%)/ 3 3 4 CH3CN (55%). Flow rate, 4 mL/min. f A ) Et3N (0.1 mol dm-3) in -3 aqueous H3PO4 (1.0 mol dm ), B ) CH3CN. Gradient: 0 min, B ) 20%, 1-5 min, B ) 45%, 5-31 min, B ) 45% (isocratic), 31-32 min, B ) 20%. Flow rate, 1.0 mL/min. g Et3N (0.1 mol dm-3) in aqueous H3PO4 (1.0 mol dm-3) (65%)/CH3CN (35%). Flow rate, 3 mL/min.

of ethanol. The reaction mixture was kept at room temperature for 30 min. Then, 1.0 mg of the appropriate PXP ligand (dissolved in 0.25 mL of ethanol) and 2.5 mg of H2BZ1 (dissolved in 0.25 mL of ethanol) were added to the reaction vial, which was heated at 100 °C for 30 min. Chromatographic characterization of the resulting Tc-99m complexes (Table 1) showed the presence of two distinct products, A and B, with relative yields in the range 80-85% and 10-15%, respectively. Overall yields ranged between 90 and 96%. Preparation of the Complexes [99mTc(N)(PNP4)(L1)] (L1 ) C1, C2). Na[99mTcO4] (50 MBq) was added to a vial containing 5.0 mg of SDH, 0.1 mg of SnCl2 (suspended in 0.1 mL of saline), 0.5 mL of saline, and 0.5 mL of ethanol. The reaction mixture was kept at room temperature for 30 min. Then, 0.25 mL of a phosphate buffer (PBS, pH ) 7.4), 0.1 mg of PNP4 (dissolved in 0.3 mL of ethanol), and 10.0 mg of H2L1 (dissolved in 0.3 mL of ethanol) were added to the reaction vial, which was heated at 100 °C for 1 h. Yield > 90%. Chromatographic characterization is reported in Table 2. Determination of Partition Coefficients (Log P). After HPLC purification, Log P values of Tc-99m complexes 1-3 were determined by measuring the activity partitioned between n-octanol (3.0 mL) and aqueous phosphate buffer (3.0 mL, 0.1 mol dm-3, pH ) 7.4) under equilibrium conditions. Results are reported in Table 2. On the contrary, due to the impossibility to attain a true equilibrium condition with complexes 4 and 5, their Log P values were not determined. Serum Stability. After HPLC purification, 100 µL of the selected Tc-99m complex were added to a propylene test tube (5 mL) containing 900 µL of rat serum or, alternatively, 900 µL of saline. The resulting mixture was incubate at 37 °C for 2 h. RCP changes in time were checked at 15, 30, 60, and 120 min by TLC. In Vitro Reaction with Glutathione (GSH) and Cysteine. A phosphate buffer (250 µL, 0.2 mol dm-3, pH ) 7.4), water (100 µL), the appropriate HPLC-purified Tc-99m complex (100 µL), and an aliquot (50 µL) of a

stock aqueous solution of GSH (0.01 mol dm-3) were mixed in a propylene test tube (5 mL), and the mixture was incubated at 37 °C for 2 h. For the blank experiment, an equal volume of water was added in place of the GSH solution. Aliquots of the resulting solutions were withdrawn at 15, 30, 60, and 120 min after incubation and analyzed by TLC chromatography. The same procedure applied above for GSH challenge was performed in two separate experiments using two different aqueous solutions of cysteine hydrochloride (0.01 and 0.001 M, respectively). Animal Studies. All animal experiments were performed in compliance with relevant national laws and with the Principles of Laboratory Animal Care (NIH Publication #85-23, revised 1985). Purified fractions of the complexes were obtained by HPLC separation. Before injection, the collected activity was further diluted with PBS (0.1 M, pH, 7.4) to give a final solution that was 10% in ethanol content. Using Tc-99m complexes of 1-3, both peaks corresponding to the two isomers A and B were recovered, and the resulting mixture was injected for biodistribution studies. Female Sprague-Dawley rats weighing 200-250 g were anesthetized with an intramuscle injection of a mixture of ketamine (80 mg kg-1) and xilazine (19 mg kg-1). A jugular vein was surgically exposed, and 100 µL (300-370 kBq) of the solution containing the radioactive complex was injected. The animals (n ) 3) were sacrificed by cervical dislocation at different times postinjection. The blood was withdrawn from the heart through a syringe immediately after the sacrifice and counted. Harvested organs were rinsed in saline, weighed, and counted in a γ-counter. Results expressed as %ID/g tissue are reported in Tables 4-8. Brain uptake at 2 min was always corrected by subctrating blood activity measured at the same time point after injection of the radiocompound 99mTc-DTPA (diethylenetriaminepentaacetic acid ) DTPA), which is unable to penetrate the intact BBB. In Vitro Binding Studies. Binding affinities of [3H]Ro151788 and [3H]-flunitrazepam were assayed as previously described with the following modifications (31, 32). Male Sprague-Dawley rats, weighing 200-250 g, were killed by decapitation and each cerebral cortex was extracted and stored at -80 °C until use. For assay, cerebral cortex was homogenized in 50 volumes (w/v) icecold assay buffer (0.050 M Tris-citrate buffer, pH ) 7.1) with an Ultra-Turrax homogenizer (Jenkel & Kunkel) at a setting of 4 for 30 s. The resulting homogenate was centrifuged at 48000 × g for 10 min with a L8-50 M/E ultracentrifuge (Beckman). The isolated pellet was resuspended in 20-vol assay buffer and further homogenized at the same settings as detailed above. The resulting homogenate was, then, centrifuged once again. The described procedure was repeated four times and the final pellet was resuspended in 20-vol assay buffer. The bindings of both [3H]-Ro151788 and [3H]-flunitrazepam were routinely assessed in triplicate (n ) 3),


Boschi et al.

Table 3. Spectroscopic Data and Elemental Analysesa for Tc-99g Complexes complex

MW (g mol-1)

FAB MS m/z [MH]+

1A

1041

1042

2A

1025

1026

3A

749

750

4

513

514

5

569

570

a

31P 1H

NMR (ppm)

NMR, δ (ppm)

7.79-6.85 (Harom; 28H), 7.22 (NHcys; 1H), 4.97 (CHcys; 1H), 3.25, 2.33 (CH2,cys; 2H), 4.34 (CH2,BZ1; AB; 2H), 4.29 (CH2,BZ1; AB; 2H), 3.19 (OCH3,PNP2; 3H), 3.50-2.15 (various CH2,PNP2; 12H) 7.54-7.21 (Harom; 8H), 7.16 (NHcys; 1H), 4.93 (CHcys; 1H), 3.30, 2.40 (CH2,cys; 2H), 4.35 (CH2,BZ1; AB; 2H), 4.30 (CH2,BZ1; AB; 2H), 3.30 (OCH3,PNP3; 12H), 3.14 (OCH3,PNP3; 3H), 3.53-1.77 (various CH2,PNP3; 36H) 7.57-7.22 (Harom; 8H), 7.29 (NHcys; 1H), 4.86 (CHcys; 1H), 3.25, 2.38 (CH2,cys; 2H), 4.40 (CH2,BZ1; AB; 2H), 4.33 (CH2,BZ1; AB; 2H), 2.60-1.85 (various CH2,PNP4; 8H), 1.94 (NCH3,PNP4; 3H), 1.73-1.65 (PCH3,PNP4; 12H) 7.67-7.27 (Harom; 5H), 4.21 (CH2C1, 2H), 2.60-1.86 (various CH2,PNP4; 8H), 2.07 (NCH3,PNP4; 3H), 1.76-1.68 (PCH3,PNP4; 12H) 7.62-7.29 (Harom; 5H), 3.98 (CHC2; 1H), 1.86-1.84 (CH2,C2, CHC2; 3H), 1.17 (CH3,C2; 6H), 2.68-1.80 (various CH2,PNP4; 8H), 2.03 (NCH3,PNP4; 3H), 1.83-1.72 (PCH3,PNP4; 12H)

IR (cm-1) ν (TctN)

22.7 (bs), 33.2 (bs)

1051

22.8 (bs), 30.4 (bs)

1053

14.3 (bs), 20.2 (bs)

1057

14.6 (bs), 20.8 (bs)

1058

15.0 (bs), 19.8 (bs)

1060

elemental analysis C51H51N5O5P2SClTc: C, 58.71 (58.78); H, 4.85 (4.89); N, 6.67 (6.72); S, 3.00 (3.07) C43H67N5O9P2SClTc: C, 50.40 (50.34); H, 6.56 (6.53); N, 6.79 (6.83); S, 3.05 (3.12) C29H39N5O4P2SClTc: C, 46.50 (46.45); H, 5.23 (5.20); N, 9.29 (9.34); S, 4.18 (4.27) C18H30N3O2P2STc: C, 42.21 (42.13); H, 5.83 (5.85); N, 8.11 (8.19); S, 6.17 (6.23) C22H38N3O2P2STc: C, 46.49 (46.42); H, 6.69 (6.67); N, 7.32 (7.32); S, 5.54 (5.62)

Theoretical values are in parentheses.

Table 4. Biodistribution of the Complex [99mTc(N)(PNP2)(BZ1)]

Table 7. Biodistribution of the Complex [99mTc(N)(PNP4)(C1)]

organ

2 min

10 min

30 min

60 min

organ

2 min

10 min

30 min

60 min

blood brain heart lung liver kidney

2.85 ( 0.32 0.11 ( 0.00 0.80 ( 0.00 1.53 ( 0.40 8.19 ( 1.66 1.37 ( 0.29

0.57 ( 0.06 0.02 ( 0.00 0.28 ( 0.02 1.01 ( 0.15 9.77 ( 0.91 1.05 ( 0.14

0.30 ( 0.13 0.01 ( 0.00 0.20 ( 0.06 0.37 ( 0.12 7.73 ( 2.09 0.93 ( 0.21

0.24 ( 0.04 0.01 ( 0.00 0.21 ( 0.00 0.39 ( 0.08 5.63 ( 0.45 1.06 ( 0.05


0.53 ( 0.03 0.02 ( 0.00 0.20 ( 0.01 0.35 ( 0.05 4.51 ( 0.59 4.35 ( 0.88

0.18 ( 0.03 0.01 ( 0.00 0.08 ( 0.01 0.18 ( 0.04 1.68 ( 0.25 0.70 ( 0.19

0.06 ( 0.00 0.00 ( 0.00 0.04 ( 0.01 0.06 ( 0.01 0.48 ( 0.10 0.20 ( 0.01

0.04 ( 0.00 0.00 ( 0.00 0.03 ( 0.00 0.09 ( 0.03 0.35 ( 0.10 0.15 ( 0.02

a Values represent % ID/g wet tissue ( standard deviations (n ) 4).


Table 5. Biodistribution of the Complex [99mTc(N)(PNP3)(BZ1)]a

Table 8. Biodistribution of the Complex [99mTc(N)(PNP4)(C2)]

organ

2 min

10 min

30 min

60 min

organ

2 min

10 min

30 min

60 min


0.90 ( 0.20 0.05 ( 0.01 0.37 ( 0.02 0.59 ( 0.07 3.67 ( 0.97 3.76 ( 0.07

0.28 ( 0.04 0.01 ( 0.00 0.11 ( 0.07 0.26 ( 0.02 1.12 ( 0.32 1.49 ( 0.46

0.11 ( 0.00 0.03 ( 0.00 0.05 ( 0.01 0.09 ( 0.01 0.05 ( 0.02 0.40 ( 0.04

0.06 ( 0.00 0.04 ( 0.00 0.03 ( 0.01 0.06 ( 0.01 0.03 ( 0.02 0.25 ( 0.03


0.83 ( 0.11 1.03 ( 0.13 0.96 ( 0.03 0.65 ( 0.09 5.98 ( 0.65 2.94 ( 0.07

0.18 ( 0.03 0.53 ( 0.02 0.22 ( 0.09 0.36 ( 0.03 1.92 ( 0.20 0.68 ( 0.18

0.09 ( 0.00 0.08 ( 0.00 0.04 ( 0.01 0.06 ( 0.01 0.87 ( 0.07 0.32 ( 0.05

0.05 ( 0.00 0.01 ( 0.00 0.02 ( 0.00 0.06 ( 0.01 0.09 ( 0.02 0.12 ( 0.02



Table 6. Biodistribution of the Complex [99mTc(N)(PNP4)(BZ1)]

subtracting the nonspecific binding from the total binding and expressed as fmol/mg of protein. The total protein content was determined by Bradford’s method (Comassie Brilliant Blue, Biorad) using human serum albumin as a standard (Kabi-vitrum). To study the effect of the free ligands and of the corresponding Tc-99g complexes 1-5 and on the specific binding of [3H]-Ro151788 and [3H]-flunitrazepam, binding inhibition experiments were conducted on isolated membranes obtained as described before. In particular, with complexes 1 and 2, only the main isomeric form A was utilized. On the contrary, with complex 3 both isomers A and B were isolated and evaluated. In a typical experiment, 100 µL of the membrane suspension and a final concentration of 1.0 × 10-9 M, the radioligand [3H]Ro151788 (or, alternatively, of [3H]-flunitrazepam), dissolved in a Tris-citrate buffer, were used. The total volume was 0.250 mL, and the tubes were incubated for 1 h at 0 °C until incubation was stopped by rapid filtration through a glass-fiber filter as described above. Each filter was washed three times with 3.0 mL of icecold buffer and radioactivity measured. Interference of β-emission from 99gTc (Eβmax ) 0.212 MeV, T1/2 ) 2.02 × 105 y) on 3H counting was significant only at the highest 99g Tc concentrations as radioactivity was determined by

organ

2 min

10 min

30 min

60 min


1.59 ( 0.09 0.09 ( 0.01 0.55 ( 0.05 1.13 ( 0.13 2.01 ( 0.18 6.58 ( 0.64

0.58 ( 0.05 0.05 ( 0.01 0.25 ( 0.03 0.51 ( 0.04 1.09 ( 0.15 4.71 ( 0.62

0.37 ( 0.04 0.02 ( 0.01 0.16 ( 0.02 0.32 ( 0.02 0.66 ( 0.07 4.16 ( 0.94

0.25 ( 0.01 0.02 ( 0.01 0.10 ( 0.02 0.19 ( 0.01 0.57 ( 0.05 4.38 ( 0.53


at 0 °C, and using 100 µL of the above membrane preparation with a concentration in the range (0.20-4.0) × 10-9 M. At the end of the incubation period, 3.0 mL of ice-cold assay buffer were added to each incubation tube, and its contents were immediately filtered under reduced pressure through a Whatman GF/B glass-fiber filter. Each filter was, then, washed three times with 3.0 mL of ice-cold buffer. Filter-bound radioactivity was measured by liquid scintillation spectrometry after addition of 3.0 mL of Aquensure (Packard), using a LS 5081 β-counter (Beckman). Nonspecific binding was defined as the binding measured in the presence of 1.0 × 10-5 M nitrazepam (FIS). Specific binding was calculated by


Bioconjugate Chem., Vol. 14, No. 6, 2003 1285

adjusting β-counter’s window to exclude 85% of 99gTc counts from the tritium channel. The 15% 99gTc spill was subtracted from the 3H counts. Results expressed as Ki values of the competing ligand (I) (Ki ) dissociation constant at equilibrium), were determined according to the Cheng-Prussoff equation Ki ) IC50/(1 + [R]/KR), where IC50 is the concentration of I inhibiting 50% of radioligand’s binding at the radioligand concentration [R]. RESULTS

Synthesis of the Ligands. The preparation of the ligand H2BZ1 (V) has been accomplished by a synthetic sequence according to the reactions showed in Scheme 1. Starting from the commercially available desmethyldiazepam (I), alkylation with ethyl bromoacetate in DMF in the presence of sodium hydride gave the intermediate II (33). This derivative was then transformed into the corresponding acid III (34) by basic hydrolysis with lithium hydroxide. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide (EDCI) promoted the coupling of III with S-trityl cysteine tert-butyl ester (35) to furnish compound IV that was finally transformed into V after removal of both trityl and Boc protecting groups by treatment with a mixture of triethylsilane (Et3SiH) and trifluoroacetic acid (CF3CO2H) in DCM (36). Preparation and Characterization of Tc-99m and Tc-99g Complexes. The Tc-99m analogues of complexes 1-5 were prepared through a simple two-step procedure involving the reaction of a nitrido Tc-99m precursor with the ligands PXP and H2L. The preliminary step of this procedure was carried out at room temperature and led to the formation of a nitrido Tc-99m precursor through the reaction of [99mTcO4]- with SDH in the presence of SnCl2 as reducing agent (37, 38). This was then followed by the simultaneous addition of the two ligands PXP and H2L that, after a short heating at 100 °C, gave rise to the final asymmetrical complexes [99mTc(N)(PXP)(L)] in high yield (>90%). In these reactions, no formation of the symmetrical complexes, [99mTc(N)(L)2], containing two identical bidentate ligands was detected. Tc-99m complexes 1-5 were characterized by chromatographic methods. Results are reported in Table 2. When L ) BZ1, both TLC and HPLC chromatography revealed that the resulting complexes formed as an isomeric pair, and that one isomer was predominant with a radiochemical yield in the range 60-85%. The formation of the two isomers was determined by the syn or anti orientation of the bioactive group of desmethyldiazepam relative to the terminal TctN group. A representative HPLC chromatogram for the Tc-99m complex 2 is reported in Figure 4. The syn and anti isomers were separated by HPLC and their chemical properties measured independently. Partition coefficients of the syn and anti isomers of Tc-99m complexes 1-3 are given in Table 2. Complexes 1-5 were prepared with the long-lived isotope Tc-99g through a simple ligand-substitution reaction of the weakly bound Cl- groups by L onto the dichloride derivative of the metal synthon [99gTc(N)(PXP)]2+ in a dichloromethane/ethanol mixture. As observed in preparations with the γ-emitting nuclide Tc99m, no formation of the symmetrical, disubstitued complexes ([99gTc(N)(L)2]) was detected also in reactions with Tc-99g. Characterization was accomplished by elemental analysis, infrared, 1H and 31P NMR spectra, and mass spectra (Table 3). Results supported the view that the complexes possess a five-coordinated geometry

Figure 4. HPLC chromatogram of the Tc-99g complex 2.

with a TctN group bound to the two phosphorus atoms of a neutral PXP ligand, and the negatively charged Oand S- atoms of one dianionic L ligand. The ligand BZ1 is coordinated through the terminal cysteine moiety (Figure 1), while the ligands C1 and C2 bind to the metal ion in the thiol form through the deprotonated SH and COOH functional groups (Figure 2). As a result, the complexes possess a vanishing charge. The chemical identity of Tc-99m complexes was assessed by comparing their HPLC chromatograms with those of the corresponding Tc-99g analogues. A close matching of HPLC retention times of Tc-99m complexes with those of the analogous Tc-99g is usually taken as a strong indication that the two series of compounds have the same chemical structure. Indeed, HPLC chromatography of Tc-99g complexes displayed a pattern similar to that observed for the analogous Tc-99m complexes. In particular, HPLC of the complexes 1-3 showed the presence of two peaks at the same retention times of the corresponding Tc-99m complexes, thus giving further support for the existence of a syn, anti isomeric pair. The origin of syn and anti isomerism is presumably due to the chirality of the central carbon atom of the coordinated cysteine ring, allowing the pendant benzodiazepine group to assume a syn or anti position with respect to the apical TctN group within the pseudo square pyramidal arrangement. However, a major difference between the two concentration scales was observed. Specifically, at the macroscopic level, one isomer was obtained almost quantitatively with complexes 1 and 2, the other isomer being produced in a very small amount (1 µM. In contrast, Ki of the compound diazepam used as a reference was 7.5 nM. DISCUSSION

The chemical structure of the ligand HBZ1 was designed by considering a plain application of the ‘bifunctional approach’. In fact, the bifunctional ligand HBZ1 can be viewed as composed of two different molecular pieces formed by a cysteine group and a diazepam derivative linked together. The cysteine group provides the chelating system for the metal, while the diazepam derivative plays the role of a biologically active moiety. A key advantage of this procedure is that the species H2BZ1 is capable to coordinate as a dianionic ligand, through the deprotonated thiol sufur atom and the carboxylic oxygen atom of the cysteine group, thus ensuring that the resulting complexes possess a neutral charge. This is a necessary requirement to promote the passage of the blood-brain barrier by a Tc-99m radiopharmaceutical. The coupling between the strong electrophilic metal fragment [Tc(N)(PXP)]2+ and the nucleophilic π-donor bidentate ligand H2BZ1 led to the formation of the neutral asymmetrical heterocomplexes 1-3. These preparations provide a definite example of the application of the ‘metal fragment approach’ based on the selective reaction of a precursor metal synthon with a ligand possessing a specific set of coordinating atoms. The strong affinity of the nucleophilic [S-,O-]-cysteine chelating system for the fragment [Tc(N)(PXP)]2+ allowed us to obtain the final products in high-yield and without the concomitant formation of the corresponding symmetrical nitride complexes containing two identical BZ1 ligands. Although complexes 1-3 were found to be stable in solution and resistant to transchelation by cysteine and GSH, complexes 4 and 5 showed a lower stability and kinetic inertness. This fact could be presumably attributed to the largest seven-membered chelating ring formed by the ligands H2C1 and H2C2 upon coordination to the metal center as compared to the shorter sixmembered ring formed by the ligand H2BZ1. Biodistribution studies in rats were carried out mostly to assess the ability of the resulting complexes to cross the BBB barrier. This requires that a suitable mechanism for trapping the activity into the cerebral tissue for a sufficient time should be operative. Biological results revealed that Tc-99m analogues of complexes 1-3 are not able to localize into the rat brain, and this behavior

Boschi et al.

was irrespective of syn and anti isomerism. Moreover, in vitro binding experiments showed that complexes 1-3 have lost almost entirely the affinity for benzodiazepine receptors. Binding affinities of both the syn and anti isomers of complex 3 were evaluated in separate experiments, but no significant difference was observed between the two isomeric forms. This fact indicates that receptor interaction does not provide a suitable brain trapping mechanism for these complexes, and therefore no clear insight into their ability to penetrate the intact BBB could be obtained. A possible explanation of this outcome could be found by considering that previous studies of NOESY NMR spectra of analogous asymmetrical heterocomplexes designed to target 5HT1A receptors revealed the existence of a weak interaction between the pendant bioactive moiety and the group attached to the nitrogen atom of the PNP ligand in both the syn and anti configurations. This interaction prevented the bioactive group to acquire the correct configuration for binding to the receptor and could be also effective in complexes discussed here (39). The biological evaluation of complexes containing the BZ1 ligand gave support to the conclusion that the bifunctional approach might not provide an efficient route to the design of benzodiazepine receptor-specific nitrido Tc-99m radiopharmaceuticals starting from the metal fragment [99mTc(N)(PXP)]2+. Therefore, the alternative ‘integrated approach’ was utilized in the attempt to lower the molecular size of the complexes while maintaining the biological activity of the starting drug. According to this method, the design of the ‘integrated complex’ was performed by seeking for a region of desmethyldiazepam that is considered nonessential for preserving its biological properties and could be, therefore, conveniently replaced by the metal-containing fragment. Previous studies (40) showed that binding affinity for this drug is associated with the region indicated by arrows in Figure 1. Thus, the phenyl ring fused with the seven-membered ring in the precursor biomolecule was completely removed to give an open system as illustrated in Figure 2. As a final step, thiol and carboxylic groups were placed at the two terminus of the resulting open system to provide the required π-donor atoms for binding to the metal fragment [99mTc(N)(PXP)]2+. Figure 2 shows the chemical representation of the final ligands (H2C1 and H2C2) designed through the application of the integrated design. These compounds easily reacted in the thiol form with the fragment [99mTc(N)(PNP4)]2+ to afford the final Tc-99m complexes 4 and 5 pictured in Figure 2. Although the integrated concept was essential in providing the route to complexes 4 and 5, these compounds cannot be strictly considered as true ‘integrated complexes’. Indeed, the molecular size and dimension of the metal fragment [99mTc(N)(PNP4)]2+ evidently do not fit within the structure of that unessential part of the H2BZ1 ligand brought aside to give the ligands H2C1 and H2C2. A classification like that of a ‘benzodiazepine mimetic’ may, therefore, appear more appropriate. Biological evaluation of complexes 4 and 5 still gave results not entirely satisfactory. Actually, though both complexes 4 and 5 possessed the correct matching of physical characteristics such as molecular weight and lipophilicity, only 5 showed a significant brain uptake at 2 min postinjection. Moreover, affinity of these complexes for benzodiazepine receptors was found to be almost negligible. These data suggest that the chemical modification brought about by the application of the integrated design has caused a strong perturbation of the starting bioactive drug. The difference in brain uptake


observed between complexes 4 and 5 is not easily interpretable considering that they differ only by a lateral isobutyl group, though the relative instability of these complexes in physiological conditions may also play a role in providing a biological trapping mechanism into the cerebral tissue. Investigation of these aspects is currently underway. CONCLUSIONS

The present study illustrates the application of the ‘metal-fragment’ approach based on the metal synthon [99mTc(N)(PXP)]2+ to the design and synthesis of a new class of small-molecule Tc-99m radiopharmaceuticals for imaging benzodiazepine receptors in the central nervous system. Results indicate that the labeling method is highly efficient and sufficiently flexible to be potentially utilized in the preparation of diagnostic tracers for a wide range of biological targets. As demonstrated by the weak brain accumulation of complex 5, there does not apparently exist any basic molecular feature of these complexes preventing their passage of the BBB barrier. However, since BBB crossing appears to be better favored for complexes having a reduced molecular size, it appears reasonable to expect that asymmetrical heterocomplexes containing the light PNP4 diphosphine ligand might find some potential application in the study of Tc-99m radiopharmaceuticals for the central nervous system. Moreover, though poor binding affinities for benzodiazepine receptors were found for the complexes described here, a careful selection of the most suitable length of the spacer connecting the metal fragment to the benzodiazepine moiety may prevent any perturbation of this latter group by the inorganic block, thus leaving the biomolecule free to interact with the receptor. ACKNOWLEDGMENT

This work was supported by Nihon Medi-Physics Co., Ltd., Tokyo, Japan. LITERATURE CITED (1) Weissman, B. A., and Raveh, L. (2003) Peripheral Benzodiazepine Receptors: on Mice and Human Brain Imaging. J. Neurochem. 84, 432-437. (2) Johnston, G. A. (2002) Medicinal Chemistry and Molecular Pharmacology of GABA(C) Receptors. Curr. Top. Med. Chem. 2, 903-913. (3) Siegel, E. (2002) Mapping of the Benzodiazepine Recognition Site on GABA(A) Receptors. Curr. Top. Med. Chem. 2, 833839. (4) Teuber, L., Watjens, F., and Jensen, L. H. (1999) Ligands fort he Benzodiazepine Binding Site - a Survey. Curr. Pharm. Des. 5, 317-343. (5) Hatazawa, J., and Shimosegawa, E. (1998) Imaging Neurochemistry of Cerebrovascular Disease with PET and SPECT. Q. J. Nucl. Med. 42, 193-198. (6) Richardson, M. P. Functional Imaging in Epilepsy. (2002) Seizure 11 (Suppl. A), 139-156. (7) Millet, P., Graf, C., Buck, A., Walder, B., and Ibanez, V. (2002) Evaluation of Tissue Models for PET and SPECT Benzodiazepine Binding Parameters. Neuroimage 17, 928942. (8) Muller, J., Saur, D., Klutmann, S., Weiller, C., Rother, J., and Clausen, M. (2002) Experience with 123I-Iomazenil SPECT in Acute Cerebral Infarction. Nucl. Med. Commun. 23, 1191-1196. (9) Bremner, J. D., Innis, R. B., White, T., Fujita, M., Silbersweig, D., Goddard, A. W., Staib, L., Stern, E., Cappiello, A., Woods, A., Baldwin, R., and Charney, D. S. (2000) SPECT

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BC034124N

Asymmetrical Nitrido Tc-99m Heterocomplexes as Potential Imaging

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