Synthesis and Biological Characterization of Novel 2

Synthesis and Biological Characterization of Novel 2-Quinolinecarboxamide Ligands of the Peripheral Benzodiazepine Receptors Bearing Technetium-99m or...
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Bioconjugate Chem. 2008, 19, 1143–1153

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Synthesis and Biological Characterization of Novel 2-Quinolinecarboxamide Ligands of the Peripheral Benzodiazepine Receptors Bearing Technetium-99m or Rhenium Andrea Cappelli,*,† Alessandra Mancini,† Francesco Sudati,‡ Salvatore Valenti,† Maurizio Anzini,† Sara Belloli,‡ Rosa Maria Moresco,‡ Mario Matarrese,‡ Mauro Vaghi,‡ Andrea Fabro,‡ Ferruccio Fazio,‡ and Salvatore Vomero† Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, Universita` degli Studi di Siena, Via A. Moro, 53100 Siena, Italy, and Istituto di Bioimmagini e Fisiologia Molecolare, CNR, Universita` di Milano/Bicocca, Istituto H. S. Raffaele, Via Olgettina 60, 20132 Milano, Italy. Received November 29, 2007; Revised Manuscript Received April 16, 2008

Potential receptor imaging agents based on Tc-99m for the in vivo visualization of the peripheral benzodiazepine receptor (PBR) have been designed on the basis of the information provided by the previously published structure-affinity relationship studies, which suggested the existence of tolerance to voluminous substituents in the receptor area interacting with 3-position of the quinoline nucleus of 2-quinolinecarboxamides 5. In the first step of the investigation, the stereoelectronic features of the above-indicated receptor area were also probed by means of 4-phenyl-3-[(1-piperazinyl)methyl]-2-quinolinecarboxamide derivatives bearing different substituents on the terminal piperazine nitrogen atom (compounds 6a-f). The structure-affinity relationship data confirmed the existence of a tolerance to bulky lipophilic substituents and stimulated the design of bifunctional ligands based on the 4-phenyl-3-[(1-piperazinyl)methyl]-2-quinolinecarboxamide moiety (compounds 6h,j,k,m). The submicromolar PBR affinity of rhenium complexes 6j,m suggests that the presence of their metal-ligand moieties with encaged rhenium is fairly compatible with the interaction with the PBR binding site. Thus, in order to obtain information on the in vivo behavior of these bifunctional ligands, 99mTc-labeled compounds 6h,k were synthesized and evaluated in preliminary biodistribution and single photon emission tomography (SPET) studies. The results suggest that both tracers do not present a clear preferential distribution in tissues rich in PBR, probably because of their molecular dimensions, which may hamper both the intracellular diffusion toward PBR and the interaction with the binding site.

INTRODUCTION The peripheral benzodiazepine receptor (PBR) is a 169-amino acid protein (18 kDa) with five trans-membrane domains localized on the mitochondrial outer membrane (1–3). PBR is functionally linked to the voltage-dependent anion channel (VDAC) and to the adenine nucleotide translocase (ANT) and might be implicated in the regulation of the opening of the mitochondrial permeability transition pore (MPTP). This receptor is highly expressed in steroidogenic tissues such as adrenal gland, but also in kidney, heart, testis, and at a lower level in the brain parenchyma, ependyma, choroid plexus, and olfactory neurons. Moreover, PBR is overexpressed in a variety of tumors (e.g., certain brain tumors, ovarian cancer, liver tumors, breast carcinoma, colorectal cancer, etc.) and its expression appears to be related to the degree of tumor malignancy (4–11). In addition, PBR is overexpressed on activated microglial cells localized in lesioned brain areas of patients with neurodegenerative or neuroinflammatory diseases like Alzheimer’s disease, and Huntington’s disease or multiple sclerosis (12–14). PK11195 (1, Figure 1) is an isoquinolinecarboxamide derivative, which was both found to bind the PBR with nanomolar affinity and is the most widely used pharmacological tool for the study of the expression and the function of PBR. Carbon11 labeled compound 1 was used for the in vivo imaging of microglial/macrophage activation present in different brain * Corresponding author. Tel.: +39 0577 234320; fax: +39 0577 234333; e-mail: [email protected]. † Universita` degli Studi di Siena. ‡ Universita` di Milano/Bicocca.

Figure 1. Structures of PBR ligands 1-4.

disorders including tumors by means of positron emission tomography (PET) (15–19). In addition, a potential role for compound 1 and more in general for PBR in the in vivo monitoring of macrophage infiltration in respiratory disorders has been recently proposed (20–22). These results have stimulated the research and development of a large number of potential PET radiotracers based on PBR ligands in different

10.1021/bc700437g CCC: $40.75  2008 American Chemical Society Published on Web 05/30/2008

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laboratories all over the world (for a recent review, see ref (23)). Among the most significant, labeled DAA1106 (2a: R ) 11CH3), and its fluoromethyl (2b: R ) 18FCH2) and fluoroethyl (2c: R ) 18FCH2CH2) derivatives were described and [18F]FEDAA1106 (2c) was proposed as a useful PET ligand for PBR which is currently used for the PBR imaging in human brain (24). Potent 2-quinolinecarboxamide PBR ligands 3a-c were 11Clabeled and the biodistribution studies suggested that these compounds are promising PET tracers for the in vivo imaging of PBR (25–27). Their evaluation in an excitotoxic model of Huntington’s disease in rats showed that benzyl derivative 3c can be considered an interesting candidate for the in vivo PET monitoring of neurodegenerative processes (28). Moreover, compound 3c served as a lead in the design of fluoroderivative 4, which shows subnanomolar PBR affinity, and was therefore considered for labeling with C-11 and F-18. The results obtained with 11C-labeled 4 in ex vivo biodistribution, inhibition, and small animal YAP-(S)PET studies are very promising (29), and the 18F-labeling of 4 is now in progress. Nevertheless, C-11 and F-18 labeling shows some limitations, since the production of these radioisotopes requires the use of an on-site cyclotron, an apparatus that is not always present in all nuclear medicine centers. On the other hand, technetium99m is the ideal diagnostic single-photon radionuclide with a 6 h half-life, 140 keV γ-radiation, no particulate radiation, and ready availability in all nuclear medicine centers by means of transportable 99Mo/99mTc generators (30, 31). These advantages explain the general use of Tc-99m-labeled compounds in routine clinical practice and, at the same time, stimulate the design and synthesis of new PBR ligands labeled with Tc-99m. The preparation of a receptor-ligand bearing Tc-99m is a difficult undertaking because the compound must satisfy a series of physicochemical requirements in order to reach and interact selectively with the target receptor. For instance, passive transport through biological membranes can be achieved only for complexes having a neutral charge and a suitable lipophilic feature. Furthermore, the molecular shape of the receptor imaging agent has to fit the binding site of the target receptor to allow the establishment of a strong and selective interaction (32). In the design of the receptor imaging agents based on Tc99m, two main strategies have been developed in recent years (32–34). The bifunctional chelate approach is based on the conjugation of the bioactive group (receptor-ligand moiety, RLM) to a metal complex through a suitable linkage. Thus, the “bifunctional ligand” can be designed as the result of the combination of a strong chelating group for the radionuclide (metal-ligand moiety, MLM) with the RLM by means of a suitable linker connecting these two molecular portions. After the radionuclide has been coordinated by the chelating system, the resulting conjugate shows RLM as an appended side chain. The design of new receptor imaging agents based on Tc99m for the in vivo visualization of the PBR could rely on the information provided by the previously published structureaffinity relationship studies, which suggested the presence of tolerance to voluminous substituents in the receptor area interacting with 3-position of the quinoline nucleus of 2-quinolinecarboxamides 5 (35, 36). Therefore, bifunctional ligands 6 (Figure 2) could be envisioned to interact with a sufficient potency with PBR given that the binding site would be capable of accommodating the MLM with the encaged radionuclide. In the first step of the investigation, the stereoelectronic features of the above-indicated receptor area was explored by means of probes 6a-f and unlabeled compounds 6j,m bearing rhenium in their MLM. Rhenium is an excellent model for the radioactive Tc-99m because it forms

Cappelli et al.

Figure 2. Design of PBR ligands 6.

equally stable complexes showing lipophilicity and square pyramidal structures very similar to those formed by technetium. In the second phase, potential receptor imaging agents 6h,k bearing Tc-99m were synthesized and evaluated in preliminary ex-vivo biodistribution and single photon emission tomography (SPET) studies.

EXPERIMENTAL PROCEDURES Chemistry. All chemicals used were of reagent grade. Yields refer to purified products and are not optimized. Melting points were determined in open capillaries on a Gallenkamp apparatus and are uncorrected. Microanalyses were carried out by means of a Perkin-Elmer 240C or a Perkin-Elmer Series II CHNS/O Analyzer 2400. Merck silica gel 60 (230-400 mesh) was used for column chromatography. Merck TLC plates, silica gel 60 F254, were used for TLC. 1H NMR spectra were recorded with a Bruker AC 200 or a Bruker DRX 400 AVANCE spectrometer in the indicated solvents (TMS as internal standard); the values of the chemical shifts are expressed in ppm and the coupling constants (J) in Hz. Mass spectra were recorded on either a Varian Saturn 3 spectrometer or a ThermoFinnigan LCQ-Deca. N-Benzyl-N-methyl-4-phenyl-3-[(piperazin-1-yl)methyl]2-quinolinecarboxamide (6a). A mixture of 7 (1.2 g, 3.0 mmol) in ethanol (40 mL) with piperazine (0.78 g, 9.0 mmol) was refluxed for 20 min under nitrogen. The solvent was removed under reduced pressure and the resulting residue was diluted with CHCl3. The organic layer was washed with water, dried over sodium sulfate, and evaporated under reduced pressure. The resulting dark residue was purified by flash chromatography with ethyl acetate-TEA-methanol (6:3:1) as the eluent to give 6a (0.81 g, yield 60%) as a light brown glassy solid. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 2.16 (m), 2.30 (m), 2.50 (m), 2.80 (m), 2.96 (s), 3.03 (s), 3.58 (br s), 4.38 (br s), 4.80 (s), 7.21-7.74 (m), 8.03 (d, J ) 8.4), 8.11 (d, J ) 8.3). MS(ESI): m/z 451 (M+H+). Anal. (C29H30N4O · H2O) C, H, N. 1,4-Bis-[[2-[(N-benzylmethylamino)carbonyl]-4-phenylquinolin-3-yl]methyl]piperazine (6b). This compound was obtained from the flash chromatography purification of compound 6a as a less polar fraction [dichloromethane-ethyl acetate (7:3) as the eluent]. Compound 6b was obtained as a white solid (0.30 g, yield 25%, mp 290-293 °C dec.). Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of different rotamers in equilibrium. For the sake of simplification the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 2.12 (br s), 2.80 (s), 2.85 (s), 2.89 (s), 2.93 (s), 3.47 (br s), 3.58 (br s), 4.22 (br s), 4.67 (s), 4.73 (s), 7.21-7.70 (m), 7.98-8.12 (m). MS(ESI): m/z 815 (M+H+). Anal. (C54H50N6O2 · H2O) C, H, N. 3-[[4-(Anilinocarbonyl)piperazin-1-yl]methyl]-N-benzyl-Nmethyl-4-phenyl-2-quinolinecarboxamide (6c). To a solution of 6a (0.10 g, 0.22 mmol) in CHCl3 (6 mL) cooled at 0-5 °C, a solution of phenylisocyanate (24 µL, 0.22 mmol) in CHCl3 (2 mL) was slowly added. The reaction mixture was stirred for 1 h at room temperature, washed with water, dried over sodium

Novel 2-Quinolinecarboxamide Ligands

sulfate, and evaporated under reduced pressure. The residue was purified by flash chromatography with dichloromethane-ethyl acetate (7:3) as the eluent to give 6c (0.082 g, yield 65%) as a pale yellow glassy solid. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 2.14 (m), 2.28 (m), 2.94 (s), 3.00 (m), 3.35 (m), 3.55 (br s), 4.35 (br s), 4.77 (s), 6.62 (s), 6.90 (m), 7.12-7.71 (m), 8.03 (d, J ) 8.3), 8.10 (d, J ) 8.4). MS(ESI): m/z 570 (M+H+). Anal. (C36H35N5O2 · 0.5H2O) C, H, N. N-Benzyl-3-[[4-[2-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)ethyl]piperazin-1-yl]methyl]-N-methyl-4-phenyl-2-quinolinecarboxamide (6d). A mixture of 6a (0.13 g, 0.29 mmol) in DMF (20 mL) with N-(2-bromoethyl)phthalimide (0.11 g, 0.43 mmol) and Na2CO3 (0.31 g, 2.9 mmol) was heated at 120 °C for 2 h. The solvent was removed under reduced pressure and the resulting residue was diluted with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate, and evaporated under reduced pressure. The residue was purified by flash chromatography with ethyl acetate as the eluent to give 6d (0.043 g, yield 24%) as a pale yellow glassy solid. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 2.02-2.45 (m), 2.62 (t, J ) 6.5), 2.94 (s), 3.01 (s), 3.40-3.87 (m), 4.36 (s), 4.78 (s), 7.18-7.85 (m), 8.02 (d, J ) 8.3), 8.10 (d, J ) 8.3). MS(ESI): m/z 624 (M+H+). Anal. (C39H37N5O3 · 0.67H2O) C, H, N. N-Benzyl-3-[[4-[3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)propyl]piperazin-1-yl]methyl]-N-methyl-4-phenyl-2-quinolinecarboxamide (6e). A mixture of 6a (0.15 g, 0.33 mmol) in ethanol (20 mL) with N-(3-bromopropyl)phthalimide (0.13 g, 0.49 mmol) and Na2CO3 (0.35 g, 3.3 mmol) was refluxed for 12 h. The solvent was removed under reduced pressure and the resulting residue was diluted with ethyl acetate. The organic layer was washed with water, dried over sodium sulfate, and evaporated under reduced pressure. Purification of the residue by flash chromatography with ethyl acetate as the eluent gave 6e as a pale yellow oil, which crystallized on standing (0.10 g, yield 47%, mp 177-179 °C). Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 1.72-2.38 (m), 2.90 (s), 2.98 (s), 3.43 (br s), 3.71 (m), 4.32 (br s), 4.75 (s), 7.20-7.76 (m), 8.01 (d, J ) 8.3), 8.09 (d, J ) 8.4). MS(ESI): m/z 638 (M+H+). Anal. (C40H39N5O3) C, H, N. N-Benzyl-3-[[4-[4-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)butyl]piperazin-1-yl]methyl]-N-methyl-4-phenyl-2-quinolinecarboxamide (6f). The title compound was prepared from 6a (0.15 g, 0.33 mmol), N-(4-bromobutyl)phthalimide (0.14 g, 0.50 mmol), and Na2CO3 (0.35 g, 3.3 mmol) following the procedure described for 6e, and was purified by flash chromatography with ethyl acetate as the eluent to obtain 6f (0.087 g, yield 40%) as a pale yellow oil. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 1.31-1.68 (m) 1.90-2.43 (m), 2.93 (s), 3.01 (s), 3.42-3.71 (m), 4.34 (br s), 4.78 (s), 7.18-7.82 (m), 8.02 (d, J ) 8.5), 8.09 (d, J ) 8.3). MS(ESI): m/z 652 (M+H+). Anal. (C41H41N5O3 · C4H8O2 · 0.67CH2Cl2) C, H, N.

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N-Benzyl-3-[[4-[N-[(benzoylthio)acetyl]glycylglycylglicyl]piperazin-1-yl]methyl]-N-methyl-4-phenyl-2quinolinecarboxamide (6g). A mixture of 8 (37–39) (0.12 g, 0.23 mmol) in acetonitrile (20 mL) with 6a (0.10 g, 0.22 mmol) was stirred at room temperature for 2 h under nitrogen. The reaction mixture was then poured into ice-water and the precipitate was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The resulting brown foam was purified by flash chromatography with ethyl acetate-methanol (85:15) as the eluent to give amide derivative 6g (0.12 g, yield 68%) as a light brown foam. Since the amide nitrogen of this compound bears two different substituents, the 1 H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 2.11 (m), 2.31 (m), 2.80 (m), 2.95 (s), 3.02 (s), 3.12 (m), 3.26 (m), 3.47 (m), 3.59 (m), 3.78 (s), 3.84 (d, J ) 4.0), 3.98 (m), 4.36 (s), 4.77 (s), 6.80-7.07 (m), 7.19 (m), 7.33-7.73 (m), 7.92 (m), 8.03 (d, J ) 8.3), 8.12 (d, J ) 8.4). MS(ESI): m/z 822 (M+Na+). Anal. (C44H45N7O6S · 1.5H2O) C, H, N. N-[3-[4-[[2-[(N-benzylmethylamino)carbonyl]-4-phenylquinolin-3-yl]methyl]piperazin-1-yl]propyl]-N-[[[2-[(triphenylmethyl)thio]ethyl]amino]carbonylmethyl]-S-(triphenylmethyl)2-aminoethanethiol (6i). A mixture of 6a (0.19 g, 0.42 mmol), 9 (40–42) (0.32 g, 0.42 mmol), Na2CO3 (0.22 g, 2.1 mmol), and NaI (0.08 g, 0.53 mmol) in dry CH3CN was refluxed for 9 h under nitrogen. The reaction mixture was cooled to room temperature and the solid was filtered off. The filtrate was evaporated under reduced pressure and the residue was purified by flash chromatography with ethyl acetate-TEA (95:5) as the eluent to give 6i (0.36 g, yield 73%) as a light brown foam. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 1.45 (m), 1.93-2.35 (m), 2.80-3.02 (m), 3.59 (br s), 4.35 (br s), 4.79 (s), 7.05-7.72 (m), 8.05 (d, J ) 8.4), 8.13 (d, J ) 8.4). MS(ESI): m/z 1169.6 (M+H+). Anal. (C76H76N6O2S2 · 2H2O) C, H, N. [N-[3-[4-[[2-[(N-benzylmethylamino)carbonyl]-4-phenylquinolin-3-yl]methyl]piperazin-1-yl]propyl]-N-[[(2-mercaptoethyl)amino]carbonylmethyl]-2-aminoethanethiolato]rhenium(V) Oxide (6j). Compound 6i (0.16 g, 0.14 mmol) was dissolved into boiling ethanol (10 mL) under nitrogen. To this a solution of SnCl2 (0.04 g, 0.18 mmol in 0.85 mL of 0.05 N HCl) was added, followed immediately by a solution of NaReO4 (0.05 g, 0.18 mmol in 0.85 mL of 0.05 N HCl). The resulting mixture was refluxed for 26 h. Subsequently, the addition of the same amounts of SnCl2 and NaReO4 was repeated twice and the reaction mixture was refluxed for additional 26 h, diluted with acetonitrile, and filtered. The filtrate was evaporated under reduced pressure and the resulting brown residue was partitioned between a saturated NaHCO3 solution and chloroform. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The resulting residue was purified by flash chromatography with ethyl acetate-TEA (95:5) as the eluent to give 6j (0.061 g, yield 49%) as a pink glassy solid. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (400 MHz, CDCl3): 0.91 (m), 1.07-1.37 (m), 1.55-2.40 (m), 2.81 (m), 2.90 (s), 2.94 (s), 2.99 (s), 3.02 (s), 3.16-3.36 (m), 3.55 (m), 3.88 (m), 4.04 (m), 4.36 (br s), 4.59 (m), 4.78 (s), 7.22-7.71 (m), 8.02 (d, J ) 7.8), 8.10 (d, J ) 8.1). MS(ESI): m/z 885 (M+H+). Anal. (C38H45N6O3ReS2 · C4H8O2) C, H, N.

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N-[[4-[[2-[(N-benzylmethylamino)carbonyl]-4-phenylquinolin-3-yl]methyl]piperazin-1-yl]carbonylmethyl]-N-[[[2-[(triphenylmethyl)thio]ethyl]amino]carbonylmethyl]-S-(triphenylmethyl)-2-aminoethanethiol (6l). To a solution of 10 (43) (0.23 g, 0.31 mmol) with 6a (0.18 g, 0.40 mmol) in dry CH2Cl2 (15 mL) cooled at 0-5 °C, HOBt (0.19 g, 1.4 mmol) was added. The resulting mixture was stirred at 0-5 °C for 30 min and a solution of EDCI (0.35 g, 1.8 mmol) in dry CH2Cl2 (7 mL) was added. The reaction mixture was stirred at room temperature for 18 h and washed in sequence with 1 M HCl, a saturated solution of NaHCO3 and H2O, dried over sodium sulfate, and evaporated under reduced pressure. The residue was purified by chromatography with ethyl acetate-petroleum ether (7:3) as the eluent to give 6l (0.17 g, yield 47%) as a white foam. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 2.03- 2.37 (m), 2.57-2.69 (m) 2.91-3.17 (m), 3.39 (br s), 3.60 (br s), 4.35 (br s), 4.77 (s), 7.07-7.72 (m), 8.04 (d, J ) 8.3), 8.12 (d, J ) 8.3). MS(ESI): m/z 1169.7 (M+H+). Anal. (C75H72N6O3S2 · 2H2O) C, H, N. [N-[[4-[[2-[(N-benzylmethylamino)carbonyl]-4-phenylquinolin-3-yl]methyl]piperazin-1-yl]carbonylmethyl]-N-[[(2-mercaptoethyl)amino]carbonylmethyl]-2-aminoethanethiolato]rhenium(V) Oxide (6m). Compound 6l (0.16 g, 0.14 mmol) was dissolved into boiling ethanol (15 mL) under nitrogen. To this a solution of SnCl2 (0.04 g, 0.18 mmol in 0.85 mL of 0.05 N HCl) was added, followed immediately by a solution of NaReO4 (0.05 g, 0.18 mmol in 0.85 mL of 0.05 N HCl). The resulting mixture was refluxed for 17 h. Subsequently, the addition of the same amounts of SnCl2 and NaReO4 was repeated once and the reaction mixture was refluxed for additional 24 h, diluted with acetonitrile, and filtered. The filtrate was evaporated under reduced pressure and the resulting brown residue was partitioned between a saturated NaHCO3 solution and chloroform. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The resulting residue was purified by flash chromatography with ethyl acetate-TEA (85:15) as the eluent to give 6m (0.012 g, yield 10%) as pink glassy solid. Since the amide nitrogen of this compound bears two different substituents, the 1H NMR spectrum shows the presence of two different rotamers in equilibrium. For the sake of simplification, the integral intensities have not been given. 1H NMR (200 MHz, CDCl3): 0.87 (m), 1.10-1.36 (m), 1.45-1.63 (m), 1.95-2.40 (m), 2.56-3.80 (m), 4.09-4.38 (m), 4.55-4.83 (m), 7.24-7.67 (m), 8.04 (d, J ) 8.5), 8.11 (d, J ) 8.3). MS(ESI): m/z 885 (M+H+). Anal. (C37H41N6O4ReS2) C, H, N. Radiosynthesis. Negatively charged complex 6h was prepared by means of the conventional SnCl2 radiolabeling procedure. Briefly, to 0.3 mg of the ligand in 0.30 mL of CH3CN and 150 µL of 1 M Na2CO3 was added 0.50 mg of SnCl2 × 2H2O dissolved in 200 µL of 0.01 N HCl containing 20 mCi of Na99mTcO4. The mixture was heated at 100 °C for 15 min and the radioligand was recovered by solid-phase extraction (SPE) on a preactivated Sep-Pak C-18 cartridge (Millipore). The SepPak was washed with water (10 mL) and eluted with ethanol (2.0 mL). The eluate was concentrated under a nitrogen stream. The residue was dissolved into saline and the resulting solution was sterilized through a sterile 0.22 µm filter (Gelman Acrodisc). High-performance liquid chromatography (HPLC) was performed with AKTA basic gradient system (GE Health Care) and relative variable-wavelength UV detector in series with radiochemical Bioscan Flow Count detector. The quality control assays of the unlabeled reference standard 6g and final radioligand 6h were performed on a reverse-phase analytical HPLC Chromolith C-18; (100 × 4.6 mm) with the

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Figure 3. Radiochemical purity of radioligand 6h (RT ) 10.22) in comparison with unlabeled reference standard 6g (RT ) 10.45).

following solvent systems: A, 0.1% TFA in water; B, CH3CN. Gradient: 0-1 min 100% A, 1-16 min 100% B; 16 min 0% B; 16-20 min 100% B. In the analysis of the 99mTc labeled compound 6h, unlabeled reference standard 6g was used for comparison (Figure 3). Neutral technetium complex 6k was prepared by thiol group deprotection and labeling by means of glucoheptonate (GH) as the transfer ligand. Briefly, 0.5 mg of ligand was deprotected in 2.5 mL of EtOH with 0.5 mL of acetone and 0.4 mL of 0.5 N HCl by heating at 100 °C for 15 min. To the deprotected ligand solution was added 0.5 mL of glucoheptonate kit previously labeled with 150-200 mCi of Na99mTcO4, and the reaction mixture was heated at 100 °C for 15 min. The radioligand was recovered by solid-phase extraction (SPE) on preactivated Sep-Pak C-18 cartridge (Millipore). The Sep-Pak was washed with ammonium acetate 0.1 M (5 mL) before eluting with ethanol (2 mL). The eluate was concentrated under a nitrogen stream. The residue was dissolved into saline and the resulting solution was sterilized through a sterile 0.22 µm filter (Gelman Acrodisc). HPLC was performed with the same system as above. Quality control evaluation of the unlabeled reference standard 6j, 99mTcGH, and final radioligand was performed on a reverse-phase analytical HPLC Alltech C-8 (250 × 4.6 mm, 5 µm) with the following solvent systems: A, 0.1 M ammonium acetate; B, CH3CN. Gradient: 0-1 min 5% B; 1-16 min 100% B; 16-19 min 100% B; 19 min 5% B; 19-25 min 5% B (Figure 4). Instant thin-layer chromatography on silica gel (ITLC-SG, Gelman Sciences) was performed by using different mobile phases. Acetone was used to determine 99mTc-GH (Rf ) 0) and the amount of free 99mTcO4 (Rf ) 1); 50% acetonitrile-water; for 99mTc colloid (Rf ) 0). In Vitro Binding Assays. Male Sprague-Dawley CD rats (Charles River Italia, Calco, CO, Italy) with body masses of 200-250 g were used. Rats were acclimatized to the new housing conditions for at least one week. They were housed six per cage under an artificial 12-h-light, 12-h-dark cycle at a constant temperature of 22 ( 2 °C and a relative humidity of 65%. They had free access to water and standard laboratory food at all times. Animal care and handling throughout the

Novel 2-Quinolinecarboxamide Ligands

Bioconjugate Chem., Vol. 19, No. 6, 2008 1147 Scheme 1a

Figure 4. Radiochemical purity of radioligand 6k (RT ) 14.96-15.34) in comparison with unlabeled reference standard 6j (RT ) 14.3). Compounds 6j,k are resolved into the corresponding rotamers.

experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC). Rats were sacrificed by decapitation and their brains were rapidly dissected into the various areas which were stored at -80 °C until the day of the assay. The binding assays were performed as described in the literature (44). The cerebral cortex was subsequently thawed and then homogenized in 50 volumes of ice-cold Dulbecco’s phosphate-buffered saline (PBS) pH 7.4 at 4 °C with a Polytron PT 10 disrupter (setting 5 for 20 s). The homogenate was centrifuged at 40 000g and 4 °C for 30 min and the resulting pellet was resuspended in the same volume of fresh buffer and recentrifuged. The new pellet was resuspended in 10 volumes of the incubation buffer (PBS) and used for the binding assay. [3H]PK11195 binding was measured in a final volume of 500 µL, consisting of 50 µL of membrane suspension (0.15-0.20 mg of protein), 50 µL of [3H]PK11195 (s.a. 85.5 Ci/mmol, New England Nuclear; final assay concentration of 1 nM), 5 µL of drug solution or solvent, 395 µL of PBS. The binding reaction was performed at 25 °C for 90 min and began with the addition of membranes. The incubation was terminated by rapid filtration through glass-fiber filters (Whatman GF/B) which had been presoaked with 0.3% polyethyleneimine and placed in a cell harvester filtration manifold (Brandel). The filters were washed five times with 4 mL of ice-cold PBS, after which filter-bound radioactivity was quantified by liquid scintillation spectrometry. Nonspecific binding was defined as binding in the presence of 10 µM of unlabeled PK11195 (Sigma). Specific binding was determined by subtracting the nonspecific from the total binding and was about 80% of the total binding. The concentration of the test compounds that inhibited [3H]ligand binding by 50% (IC50) was determined by means of Jandel Sigmaplot (45) program with 6 to 10 concentrations of the displacers, each performed in triplicate. Kinetic Experiments. Animals. Male CD-1 mice (35-40 g; Charles River, Italy) were used for biodistribution, smallanimal SPET imaging, and inhibition studies. Animal experiments were approved by the Ethical Committee for animal care of the San Raffaele Hospital and carried out in agreement with the Italian and EEC recommendations for the care and use of laboratory animals. Biodistribution. All animal group (n ) 3) received an intraperitoneal injection of 0.74-0.92 MBq (20-25 µCi) of [99mTc]6h or [99mTc]6k. The mice were killed by cervical dislocation at 30, 60, and 240 min after intraperitoneal injection of the radiotracer in 200 µL saline vehicle. Blood samples were collected by cardiac puncture. The tissue samples were weighed and their radioactivity was determined in an automated γ-counter

a Reagents: (i) piperazine, EtOH; (ii) phenylisocianate, CHCl3 or N-(2-bromoethyl)phthalimide, Na2CO3, DMF or N-(3-bromopropyl)phthalimide, Na2CO3, EtOH or N-(4-bromobutyl)phthalimide, Na2CO3, EtOH.

(1282 Compugamma CS; Pharmacia/LKB Nuclear Inc.) and expressed as the percentage of injected dose per gram of tissue (%ID/g). Small Animal YAP-(S)PET Studies. The kinetics of biodistribution was further explored at later times using a dedicated small animal SPET scanner YAP-(S)PET II (ISE, Pisa, Italy). This small animal tomography is able to perform both PET and SPET modalities. It is made up of four detector heads composed each of a 4 × 4 cm2 of YAlO3:Ce (YAP:Ce) matrix of 27 × 27 elements, 1.5 × 1.5 × 20 mm3 each, directly coupled to a PSPMT (Hamamatsu R2486). The four modules are positioned on a rotating gantry; the opposite detectors are in time coincidence when used in SPET mode. For both PET and SPET modalities, the system operates in 3-D data acquisition mode with an axial field of view (FOV) of 4 cm and a transaxial FOV diameter of 4 cm, providing a set of 27 slices of 1.5 mm thickness. The switching to the SPET modality is made by replacing the tungsten septum (used in PET for shielding the scintillators from the background outside the FOV) with a highresolution parallel hole, lead collimator (0.6 mm, 0.15 mm septum) in front of each crystal. The default acquisition energy window is 50-850 keV. Compounds [99mTc]6h and [99mTc]6k were intravenously administered in two CD-1 mice. The animals were scanned in SPET modality at 1, 6, and 24 h after the injection of 3.26 and 2.61 mCi (120.6 and 96.6 MBq), respectively. Each mouse was anesthetized with 1.7% tribromoethanol solution (10 µL/g of weight, i.p.) before scanning and positioned supine on the tomograph bed, with the thorax centered in the FOV. The study was achieved acquiring two bed positions in 1 h approximately. Furthermore, a 30 min brain acquisition was performed 6 h after the injection for compound [99mTc]6h and 1 h after the injection for compound [99mTc]6k. 3D data were reconstructed by the EM (Expectation Maximization) algorithm (46). Inhibition Studies. The specificity of [99mTc]6k uptake was studied in mice pretreated with PK11195 (5 mg/kg, dissolved

1148 Bioconjugate Chem., Vol. 19, No. 6, 2008 Scheme 2a

Cappelli et al. Scheme 4a

a Reagents: (i) 6a, HOBt, EDCI, CH2Cl2; (ii) NaReO4, SnCl2 × 2H2O, 0.05 N HCl, EtOH.

Table 1. PBR Binding Affinities of Compounds 3c, 5a,b, and 6a-g,j,m a Reagents: (i) CH3CN; (ii) Na99mTcO4, SnCl2 × 2H2O, 0.01 N HCl, Na2CO3, CH3CN.

Scheme 3a

a Reagents: (i) 6a, NaI, Na2CO3, CH3CN; (ii) for compound 6j: NaReO4, SnCl2×2H2O, 0.05 N HCl, EtOH; for compound 6k: 0.5 N HCl, EtOH, CH3COCH3, GH kit labelled with Na99mTcO4.

into DMSO-EtOH 1:1, i.p., n ) 3) or with a vehicle (i.p., n ) 3) injected 60 min before the radioligand administration. The inhibitory effect of unlabeled PK11195 was evaluated 4 h after the tracer injection on the basis of our previous experience and was compared to the preinjection of the vehicle. After this time, the mice were sacrificed by cervical dislocation and processed as described above for the biodistribution to obtain the %ID/g for peripheral tissues derived from vehicle or PK11195 injected mice.

compd

R

R′

3c 5a 5b 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 1

C2H5 CH2C6H5 H See Scheme 1 CONH-C6H5 CH2CH2-phthalimido CH2CH2CH2-phthalimido CH2CH2CH2CH2-phthalimido (COCH2NH)3COCH2SCOC6H5 see Scheme 2 see Scheme 3 see Scheme 3 (M ) Re) see Scheme 3 (M ) Tc-99m) see Scheme 4 see Scheme 4

C2H5 C2H5

IC50 (nM) ( SEMa 2.1-4.6b 12b 13b 301 ( 29 70 ( 8.5 24 ( 2.6 37 ( 3.3 22 ( 1.9 36 ( 2.8 209 ( 26 NTc NTc 127 ( 15 NTc NTc 187 ( 19 2.5 ( 0.4

a Each value is the mean ( SEM of 3 determinations and represents the concentration giving half the maximum inhibition of [3H]1 (final concentration 1 nM) specific binding to rat cortical membranes. b See ref 36. c NT: not tested.

RESULTS Chemistry. The PBR probes 6a-f were synthesized from the previously described chloromethyl derivative 7 (36) as depicted in Scheme 1. Reaction of 7 with a large excess of piperazine gave piperazinyl derivative 6a along with a small amount of double alkylation product 6b, which was separated by flash chromatography. Phenylurea derivative 6c was prepared by reaction of 6a with phenylisocyanate, while phthalimidoalkyl derivatives 6d-f were synthesized by alkylation of 6a with the appropriate N-(bromoalkyl)phthalimides in the presence of sodium carbonate as the base.

Novel 2-Quinolinecarboxamide Ligands

Bioconjugate Chem., Vol. 19, No. 6, 2008 1149

Figure 5. Comparison of tissue distribution of 6h,k in CD-1 mouse 60 min after the radiotracer injection. The column where the error is not indicated was obtained with two mice. Table 2. Tissue Distribution of [99mTc]6h in CD-1 Mousea

Table 3. Tissue Distribution of [99mTc]6k in CD-1 Mousea

tissue

30 min

60 min

240 min

tissue

30 min

60 min

240 min

blood lung diaphragm liver intestine stomach spleen kidney muscle bone

1.65b 0.46 ( 0.12 7.26 ( 0.60 4.84 ( 0.78 0.52 ( 0.21 0.53 ( 0.10 0.69 ( 0.44 0.49 ( 0.11 0.61 ( 0.21 1.43 ( 0.53

3.52 ( 0.67 0.71 ( 0.29 6.11 ( 2.31 6.12 ( 0.94 9.52 ( 0.76 1.57 ( 0.6 1.02 ( 0.44 0.93 ( 0.22 1.08 ( 0.38 2.82 ( 1.09

0.23b 0.69b 4.20 ( 1.67 2.62 ( 1.35 3.87 ( 1.47 0.41 ( 0.24 0.49 ( 0.13 0.40 ( 0.25 0.71 ( 0.20 0.61b

blood lung diaphragm liver intestine stomach spleen kidney muscle bone

1.08 ( 0.40 1.19 ( 0.24 4.74 ( 1.3 3.48 ( 1.65 2.26 ( 0.83 1.72 ( 0.57 2.07 ( 1.32 1.28b 1.04 ( 0.75 1.18 ( 0.32

5.25b 1.16 ( 0.23 5.12 ( 0.55 5.14 ( 0.20 4.71 ( 0.77 3.77 ( 0.90 3.85 ( 0.88 2.13 ( 0.69 2.28 ( 0.01 1.43 ( 0.53

4.44 ( 1.37 1.05 ( 0.26 7.92 ( 1.35 4.80 ( 0.45 3.06 ( 1.35 2.13 ( 0.32 3.43 ( 0.72 1.50 ( 0.10 0.89 ( 0.21 1.56 ( 0.22

a Radioactivity concentration is expressed as % of injected dose per gram of tissue (%ID/g). Values are expressed as mean ( SD of three rats for each time point. b The corresponding values were obtained with two mice.

Compound 6g was obtained by conjugation of 6a with S-benzoylmercaptoacetylglycylglycylglycine(S-Bz-MAG3,Scheme 2) via activated ester 8 (37–39). Negatively charged complex 6h was prepared by means of conventional radiolabeling procedure with 99mTc (see Experimental Procedures). Cross-coupling reaction of synthon 9 (40–42) with piperazinylderivative 6a provided 6i (Scheme 3), which underwent rhenium incorporation with sodium perrhenate under reductive conditions (SnCl2) in ethanol to give the neutral complex 6j. On the other hand, neutral technetium complex 6k was prepared by employing glucoheptonate (GH) as the transfer ligand (see Experimental Procedures). Finally, neutral rhenium complex 6m (showing the N2S2 MLM of 6j and the carbonylmethyl spacer of 6 h) was prepared as shown in Scheme 4. Acid 10 was synthesized as described in the literature (43) and conjugated with the piperazinyl derivative 6a in the presence of HOBt and EDCI to afford 6l. Neutral complex 6m was obtained in low yield from 6l by means of the procedure shown above. Binding Studies. Compounds 6a-g,j,m were tested for their potential activity in inhibiting the specific binding of [3H]1 to rat cortical membrane in comparison with reference compound 1. The results of the binding studies are shown in Table 1, along with the previously published PBR affinities of 2-quinolinecarboxamide derivatives 5a,b that are included for comparison. The structure-affinity relationship (SAFIR) analysis of the compounds designed to probe the receptor tolerance to bulky substituent shows that the introduction of the piperazine substituent of compound 6a produces a significant decrease in PBR affinity. The comparison between this result and that obtained with previously published N,N-diethylderivative 5a suggests that the affinity decrease can be attributed to the presence of the second basic nitrogen atom (the terminal piperazine nitrogen), which confers to compound 6a hydrophilic characters inappropriate for the interaction with the PBR binding site. This assumption is supported by the affinity shown by phenylurea derivative 6c, which shows an IC50 value similar to that shown by compounds 5a,b.

a Radioactivity concentration is expressed as % of injected dose per gram of tissue (%ID/g). Values are expressed as mean ( SD of three rats for each time point. b The corresponding values were obtained with two mice.

Interestingly, the replacement of the -CONHPh group of phenylurea derivative 6c with a phthalimidoethyl one of 6d appears to be well-tolerated by the PBR binding site. Furthermore, the nanomolar PBR affinity shown by 6d appears to be stable to the elongation of the spacer between the terminal piperazine nitrogen and the phthalimide moiety up to three methylene unities (compound 6e) or four methylene unities (6f). These results, taken together with the nanomolar affinity shown by the symmetrically substituted piperazine derivative 6b, demonstrate that the PBR binding site is capable of accommodating a large variety of molecules based on the 4-phenylquinolin-2-carboxamide scaffold. Thus, the results of the first phase of the investigation confirmed the PBR tolerance to bulky substituents and gave impulse to the development of the second phase. In the design of the receptor imaging agents based on Tc99m, the physicochemical characteristics of the final complex were taken into consideration. Thus, neutral 6j,k,m and negatively charged 6h complexes were prepared in order to evaluate the difference in the biological properties. Protected precursor 6g and cold neutral rhenium complexes 6j,m were studied in the same binding system described for the other ligands belonging to the same class. The submicromolar PBR affinity shown by 6j,m (IC50 values of 127 and 187 nM, respectively) suggests that their MLMs with encaged rhenium interact with PBR binding site less effectively than the phthalimido probe of compounds 6d-f (IC50 values 22-37 nM). However, the reasonable affinity of rhenium complexes 6j,m and the fact that rhenium has been described to show virtually identical structural properties in its complexes compared to technetium (31) motivated the evaluation of technetium-99m labeled complexes 6h,k in order to obtain information on the in vivo behavior of these bifunctional ligands.

KINETIC STUDIES Biodistribution. Exvivo biodistribution studies in mice indicate that, after intraperitoneal injection, radiotracer

1150 Bioconjugate Chem., Vol. 19, No. 6, 2008

Cappelli et al.

Figure 6. YAP-(S)PET II images of [99mTc]6h distribution in one CD-1 mouse at 1 (a), 6 (b), and 24 h (c) after the i.v. injection of 3.26 mCi of the radiotracer. (a) 1 h. Transaxial view of 3D-rendering reconstruction of whole body acquisition (two beds in present count modality). (b) 6 h. Top, transaxial view of 3D-rendering reconstruction of brain (one bed in present time modality); bottom, transaxial view of 3D-rendering reconstruction of whole body acquisition (two beds in present count modality). (c) 24 h. Transaxial view of 3D-rendering reconstruction of whole body acquisition (two beds in present count modality).

Figure 7. YAP-(S)PET II images of [99mTc]6k distribution in one CD-1 mouse acquired at several time points after the i.v. injection of 2.61 mCi of the radioligand. (a) 1 h. Top, transaxial view of 3D-rendering reconstruction of brain and thorax (one bed in present time modality); bottom, transaxial view of 3D-rendering reconstruction of the abdomen (one bed in present time modality). (b) 6 h. Transaxial view of 3D-rendering reconstruction of whole body acquisition (two beds in present count modality). (c) 24 h. Transaxial view of 3D-rendering reconstruction of whole body acquisition (two beds in present count modality).

[99mTc]6h and [99mTc]6k preferentially accumulate in liver, intestine, and stomach. Similar results are described in the literature for the biodistribution of somatostatin analogue RC160 labeled with 188Re- or 99mTc-MAG3 (39). However, differently from the negatively charged complex 6h, neutral 6k accumulates also in two organs expressing PBR-like spleen and kidney (Figure 5, Tables 2 and 3) although radioactivity was lower than that measured in blood. Maximum levels of radioactivity were observed 60 min after injection of both tracers. SPET Studies. SPET biodistribution profiles of 6h and 6k are shown in Figures 6 and 7. One hour after injection, radioactivity preferentially accumulated in the liver, gallbladder, small intestine, urinary bladder, and spleen, although this last organ was better visualized in the animal injected with neutral complex 6k (Figure 7). At this time, accumulation of radioactivity was observed in the stomach of the animal injected with 6h and a small signal of uptake appeared in the lung of the animal injected with 6k. Six hours after the

injection of both radiotracers, the majority of the activity shifted to the first portion of the small intestine especially in the case of compound 6h. Finally after 24 h, radioactivity was still present in the liver and intestine for 6h and in liver and urinary bladder for 6k. For both compounds, we failed to observe a significant radioactivity accumulation in the heart and brain. Inhibition Studies. The specificity of [99mTc]6k in vivo binding to PBR was evaluated in mice pretreated with PK11195 (5 mg/kg, i.p., n ) 3) or with the vehicle (i.p., n ) 3), 1 h before the radioligand administration. The results of the inhibition experiments 4 h after [99mTc]6k injection are shown in Figure 8. The only significant effect of PK11195 preadministration was observed in the lung, while no significant effects were observed in the remaining tissues.

DISCUSSION AND CONCLUSIONS The exploration of the PBR binding site by means of 3-substituted-2-quinolinecarboxamide derivatives 6a-f con-

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Bioconjugate Chem., Vol. 19, No. 6, 2008 1151

LITERATURE CITED

Figure 8. Effect of PK11195 pretreatment on [99mTc]6k radioactivity uptake in peripheral tissues of mice measured 4 h after [99mTc]6k injection. The radioactivity concentration is expressed as % of the injected dose per gram of tissue (%ID/g); 5 mg/kg of PK11195 (pretreated, black) or vehicle (basal, white) were administered 1 h before [99mTc]6k injection.

firmed the existence of a tolerance to bulky lipophilic substituents and stimulated the design and synthesis of bifunctional ligands 6h,j,k,m. The submicromolar PBR affinity shown by cold rhenium complexes 6j,m encouraged the preparation of technetium-99m labeled compounds 6h,k and their evaluation in ex vivo biodistribution and in small animal SPET studies. The results obtained indicate that both tracers do not present a clear preferential distribution in high PBR expressing tissues. In fact, none of them was taken up by the heart, and only neutral complex 6k displayed activity in spleen, kidney, and lung. In addition, these levels were lower or comparable to those observed in blood and 6k uptake appeared to be inhibited by unlabeled PK11195 only in lung. A possible explanation is the high molecular weight of these tracers that may hamper transmembrane diffusion and the reaching of the intracellular target receptor. However, compounds 6h,k show very similar shapes and dimensions (i.e., MW is ca. 800), but different properties in both the spacer and the MLM. In particular, compound 6h shows the contemporary presence of a negative charge in the MLM and a basic nitrogen atom in the piperazine moiety. The protonation in the physiological environment is likely to produces a zwitterionic character in this MAG3 derivative that is not present in 6k and may play a significant role in the membrane passage and, as a consequence, in the biodistribution features. Therefore, 6h can be ultimately considered a negative control capable of highlighting the relatively favorable properties of 6k. On the basis of the above results, we believe that negatively charged complex 6h has inadequate kinetic properties to be further explored as a PBR ligand, while additional experiments on selected animal models are required to establish the potential usefulness in tumor imaging or in the monitoring of lung inflammation of neutral complex 6k based on the MLM present in technepine, fluoratec, and other interesting tracers (32, 41, 42).

ACKNOWLEDGMENT The authors are grateful to Prof. Stefania D’Agata D’Ottavi for the careful reading of the manuscript and to Prof. Giovanni Biggio and Prof. Alessandra Concas for the binding studies performed on compounds 6a-g,j,m. This work was financially supported by Ministero dell’Universita` e della Ricerca - PRIN (Programmi di ricerca di Rilevante Interesse Nazionale) and by EMIL (European Molecular Imaging Laboratory), Sixth European Program, Project No: LSHC-CT-2004-503569. Supporting Information Available: Analytical data for compounds 6a-g,i,j,l,m. This material is available free of charge via the Internet at http://pubs.acs.org.

(1) Papadopoulos, V., Baraldi, M., Guilarte, T. R., Knudsen, T. B., Lacape`re, J.-J., Lindemann, P., Norenberg, M. D., Nutt, D., Weizman, A., Zhang, M.-R., and Gavish, M. (2006) Translocator protein (18 kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol. Sci. 27, 402–409. (2) Gavish, M., Katz, Y., Bar-Ami, S., and Weizman, R. (1992) Biochemical, physiological, and pathological aspects of the peripheral benzodiazepine receptor. J. Neurochem. 58, 1589– 1601. (3) Beurdeley-Thomas, A., Miccoli, L., Oudard, S., Dutrillaux, B., and Poupon, M. F. (2000) The peripheral benzodiazepine receptors: a review. J. Neurooncol. 46, 45–56. (4) Starosta-Rubinstein, S., Ciliax, B. J., Penney, J. B., McKeever, P., and Young, A. B. (1987) Imaging of a glioma using peripheral benzodiazepine receptor ligands. Proc. Natl. Acad. Sci. U.S.A. 84, 891–895. (5) Black, K. L., Ikezaki, K., and Toga, A. W. (1989) Imaging of brain tumors using peripheral benzodiazepine receptor ligands. J. Neurosurg. 71, 113–118. (6) Black, K. L., Ikezaki, K., Santori, E., Becker, D. P., and Vinters, H. V. (1990) Specific high-affinity binding of peripheral benzodiazepine receptor ligands to brain tumors in rat and man. Cancer 65, 93–97. (7) Miettinen, H., Kononen, J., Haapasalo, H., Helen, P., Sallinen, P., Harjuntausta, T., Helin, H., and Alho, H. (1995) Expression of peripheral-type benzodiazepine receptor and diazepam binding inhibitor in human astrocytomas: relationship to cell proliferation. Cancer Res. 55, 2691–2695. (8) Batra, S., and Iosif, C. S. (1998) Elevated concentrations of mitochondrial peripheral benzodiazepine receptors in ovarian tumors. Int. J. Oncol. 12, 1295–1298. (9) Venturini, I., Alho, H., Podkletnova, I., Corsi, L., Rybnikova, E., Pellicci, R., Baraldi, M., Pelto-Huikko, M., Helen, P., and Zeneroli, M. L. (1999) Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver. Life Sci. 65, 2223–2231. (10) Hardwick, M., Fertikh, D., Culty, M., Li, H., Vidic, B., and Papadopoulos, V. (1999) Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell agressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res. 59, 831–842. (11) Maaser, K., Grabowski, P., Sutter, A. P., Hopfner, M., Foss, H. D., Stein, H., Berger, G., Gavish, M., Zeitz, M., and Scherubl, H. (2002) Overexpression of the peripheral benzodiazepine receptor is a relevant prognostic factor in stage III colorectal cancer. Clin. Cancer Res. 8, 3205–3209. (12) Diorio, D., Welner, S. A., Butterworth, R. F., Meaney, M. J., and Suranyi-Cadotte, B. E. (1991) Peripheral benzodiazepine binding sites in Alzheimer’s disease frontal and temporal cortex. Neurobiol. Aging 12, 255–258. (13) Vowinckel, E., Reutens, D., Becher, B., Verge, G., Evans, A., Owens, T., and Antel, J. P. (1997) PK11195 binding to the peripheral benzodiazepine receptor as a marker of microglia activation in multiple sclerosis and experimental autoimmune encephalomyelitis. J. Neurosci. Res. 50, 345–353. (14) Schoemaker, H., Morelli, M., and Deshmukh, P. (1982) [3H]Ro 5-4864 benzodiazepine binding in the kainate lesioned striatum and Huntington’s diseased basal ganglia. Brain Res. 248, 396–401. (15) Pike, V. W., Halldin, C., Crouzel, C., Barre`, L., Nutt, D. J., Osman, S., Shah, F., Turton, D. R., and Waters, S. L. (1993) Radioligands for PET studies of central benzodiazepine receptors and PK (peripheral benzodiazepine) binding sites - Current status. Nucl. Med. Biol. 20, 503–525. (16) Junck, L., Olson, J. M., Ciliax, B. J., Koeppe, R. A., Watkins, G. L., Jewett, D. M., McKeever, P. E., Wieland, D. M., Kilbourn, M. R., Starosta-Rubinstein, S., Mancini, W. R., Kuhl, D. E.,

1152 Bioconjugate Chem., Vol. 19, No. 6, 2008 Greenberg, H. S., and Youngf, A. B. (1989) PET imaging of human gliomas with ligands for the peripheral benzodiazepine binding site. Ann. Neurol. 26, 752–758. (17) Pappata, S., Cornu, P., Samson, Y., Prenant, C., Benavides, J., Scatton, B., Crouzel, C., Hauw, J. J., and Syrota, A. (1991) PET study of carbon-11-PK 11195 binding to peripheral type benzodiazepine sites in glioblastoma: a case report. J. Nucl. Med. 32, 1608–1610. (18) Junck, L., Jewett, D.M., Kilbourn, M. R., Young, A. B., and Kuhl, D. E. (1990) PET imaging of cerebral infarcts using a ligand for the peripheral benzodiazepine binding site. Neurology 40 (1), 265. (19) Charbonneau, P., Syrota, A., Crouzel, C., Valois, J. M., Prenant, C., and Crouzel, M. (1986) Peripheral-type benzodiazepine receptors in the living heart characterized by positron emission tomography. Circulation 73, 476–483. (20) Hardwick, M. J., Chen, M. K., Baidoo, K., Pomper, M. G., and Guilarte, T. R. (2005) In vivo imaging of peripheral benzodiazepine receptors in mouse lungs: a biomarker of inflammation. Mol. Imaging 4, 432–438. (21) Audi, S. H., Dawson, C. A., Ahlf, S. B., and Roerig, D. L. (2002) Lung tissue mitochondrial benzodiazepine receptors increase in a model of pulmonary inflammation. Lung 180, 241– 250. (22) Jones, H. A., Valind, S. O., Clark, I. C., Bolden, G. E., Krausz, T., Schofield, J. B., Boobis, A. R., and Haslett, C. (2002) Kinetics of lung macrophages monitored in vivo following particulate challenge in rabbits. Toxicol. Appl. Pharmacol. 183, 46–54. (23) Venneti, S., Lopresti, B. J., and Wiley, C. A. (2006) The peripheral benzodiazepine receptor (translocator protein 18 kDa) in microglia: from pathology to imaging. Prog. Neurobiol. 80, 308–322. (24) Zhang, M. R., Maeda, J., Ogawa, M., Noguchi, J., Ito, T., Yoshida, Y., Okauchi, T., Obayashi, S., Suhara, T., and Suzuki, K. (2004) Development of a new radioligand, N-(5fluoro-2phenoxyphenyl)-N-(2-[18F]fluoroethyl-5-methoxybenzyl)acetamide, for PET imaging of peripheral benzodiazepine receptor in primate brain. J. Med. Chem. 47, 2228–2235. (25) Matarrese, M., Moresco, R. M., Cappelli, A., Anzini, M., Vomero, S., Simonelli, P., Verza, E., Magni, F., Sudati, F., Soloviev, D., Todde, S., Carpinelli, A., Galli Kienle, M., and Fazio, F. (2001) Labeling and evaluation of N-[11C]methylated quinoline-2-carboxamides as potential radioligands for visualization of peripheral benzodiazepine receptors. J. Med. Chem. 44, 579–585. (26) Matarrese, M., Soloviev, D., Cappelli, A., Todde, S., Moresco, R. M., Anzini, M., Vomero, S., Sudati, F., Carpinelli, A., Perugini, F., Galli Kienle, M., and Fazio, F. (1999) Synthesis of the novel [11C]-labelled quinoline carboxamides: analogues of PK-11195 as putative radioligands for PET studies of peripheral type benzodiazepine receptors. J. Labelled Cpd. Radiopharm. 42, S397-S399. (27) Cappelli, A., Anzini, M., Vomero, S., De Benedetti, P. G., Menziani, M. C., Giorgi, G., and Manzoni, C. (1997) Mapping the peripheral benzodiazepine receptor binding site by conformationally restrained derivatives of 1-(2-chlorophenyl)-N-methylN-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195). J. Med. Chem. 40, 2910–2921. (28) Belloli, S., Moresco, R. M., Matarrese, M., Biella, G., Sanvito, F., Simonelli, P., Turolla, E., Olivieri, S., Popoli, P., Cappelli, A., Vomero, S., Galli-Kienle, M., and Fazio, F. (2004) Evaluation of three quinoline-2-carboxamide derivatives as potential radioligands for the in vivo PET imaging of neurodegeneration. Neurochem. Int. 44, 433–440. (29) Cappelli, A., Matarrese, M., Moresco, R. M., Valenti, S., Anzini, M., Vomero, S., Turolla, E., Belloli, S., Simonelli, P., Filannino, M. A., Lecchi, M., and Fazio, F. (2006) Synthesis, labeling, and biological evaluation of halogenated quinoline-2carboxamides as potential radioligands for visualization of peripheral benzodiazepine receptors. Bioorg. Med. Chem. 14, 4055–4066.

Cappelli et al. (30) Schwochau, K. (2000) Technetium. Chemistry and radiopharmaceutical applications; Wiley-VCH, Weinheim, Germany. (31) Fritzberg, A. R., Abrams, P. G., Beaumier, P. L., Kasina, S., Morgan, A. C., Rao, T. N., Sanderson, J. A., Srinivasan, A., Wilbur, D. S., and Vanderheyden, J. L. (1988) Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent. Proc. Natl. Acad. Sci. U.S.A. 85, 4025–4029. (32) Hom, R. K., and Katzenellenbogen, J. A. (1997) Technetium99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results. Nucl. Med. Biol. 24, 485–498. (33) Jurisson, S. S., and Lydon, J. D. (1999) Potential technetium small molecule radiopharmaceuticals. Chem. ReV. 99, 2205– 2215. (34) Chi, D. Y., Neil, J. P., Anderson, C. J., Welch, M. J., and Katzenellenbogen, J. A. (1994) Homodimeric and heterodimeric bis(aminothiol) oxometal complexes with rhenium(V) and technetium(V). Control of heterodimeric complex formation and an approach to metal complexes that mimic steroid hormones. J. Med. Chem. 37, 928–937. (35) Anzini, M., Cappelli, A., Vomero, S., Seeber, M., Menziani, M. C., Langer, T., Hagen, B., Manzoni, C., and Bourguignon, J. J. (2001) Mapping and fitting the peripheral benzodiazepine receptor binding site by carboxamide derivatives. Comparison of different approaches to quantitative ligand-receptor interaction modeling. J. Med. Chem. 44, 1134–1150. (36) Cappelli, A., Pericot Mohr, G., Gallelli, A., Giuliani, G., Anzini, M., Vomero, S., Fresta, M., Porcu, P., Maciocco, E., Concas, A., Biggio, G., and Donati, A. (2003) Structureactivity relationships in carboxamide derivatives based on the targeted delivery of radionuclides and boron atoms by means of peripheral benzodiazepine receptor ligands. J. Med. Chem. 46, 3568–3571. (37) Fritzberg, A. R., Kasina, S., Vanderheyden, J. L., and Srinivasan, A. ( 1988) Metal-radionuclide-labeled proteins and glycoproteins and their preparation for diagnosis and therapyEur. Pat. Appl. EP284071A2 CA 111:53446. (38) Grummon, G., Rajagopalan, R., Palenik, G. J., Kaziol, A. E., and Nosco, D. L. (1995) Synthesis, characterization and crystal structures of technetium (V)-oxo complexes useful in nuclear medicine. 1. Complexes of mercaptoacetylglycylglycylglycine (MAG3) and its methyl ester derivative (MAG3OMe). Inorg. Chem. 34, 1764–1772. (39) Guhlke, S., Schaffland, A., Zamora, P. O., Sartor, J., Diekmann, D., Bender, H., Knapp, F. F., and Biersack, H. J. (1998) 188Re- and 99m Tc-MAG3 as prosthetic groups for labeling amines and peptides: approaches with pre- and postconjugate labeling. Nuc. Med. Biol. 25, 621–631. (40) O’Neil, J. P., Wilson, S. R., and Katzenellenbogen, J. A. (1994) Preparation and structural characterization of monoaminemonoamide bis(thiol) oxo complexes of technetium (V) and rhenium (V). Inorg. Chem. 33, 319–323. (41) Meltzer, P. C., Blundell, P., Jones, A. G., Mahmood, A., Garada, B., Zimmerman, R. E., Davison, A., Holman, B. L., and Madras, B. K. (1997) A technetium-99m SPECT imaging agent which targets the dopamine transporter in primate brain. J. Med. Chem. 40, 1835–1844. (42) Meltzer, P. C., Blundell, P., Zona, T., Yang, L., Huang, H., Bonab, A. A., Livini, E., Fischman, A., and Madras, B. K. (2003) A second-generation 99m technetium single photon emission computed tomography agent that provides in vivo images of the dopamine transporter in primate brain. J. Med. Chem. 46, 3483– 3496. (43) Li, G., Ma, B., Missert, J. R., Grossman, Z. D., and Pandey, R. K. (1999) Synthesis of N2S2 conjugates of the highly specific mitochondrial diazepam binding inhibitor (DBI) receptor complexes. Heterocycles 51, 2855–2860. (44) Trapani, G., Laquintana, V., Denora, N., Trapani, A., Lepedota, A., Latrofa, A., Franco, M., Serra, M., Pisu, M. G., Floris, I., Sanna, E., Biggio, G., and Liso, G. (2005) Structure-

Novel 2-Quinolinecarboxamide Ligands activity relationships and effects on neuroactive steroid synthesis in a series of 2-phenylimidazo[1,2-a]pyridineacetamide peripheral benzodiazepine receptor ligands. J. Med. Chem. 48292–305. (45) Sigma Plot, Jandel Scientific Graphing Software for Windows, San Raphael, CA, USA, 1995.

Bioconjugate Chem., Vol. 19, No. 6, 2008 1153 (46) Motta, A., Damiani, C., Del Guerra, A., Di Domenico, G., and Zavattini, G. (2002) Use of a fast EM algorithm for 3D image reconstruction with the YAP-PET tomograph. Comput. Med. Imaging Graphics 26, 293–302. BC700437G