Synthesis and evaluation of radiolabeled piperazine derivatives of vesamicol as SPECT agents for cholinergic neurons

Nuclear Medicine and Biology 28 (2001) 251–260

Synthesis and evaluation of radiolabeled piperazine derivatives of vesamicol as SPECT agents for cholinergic neurons Kazunori Bandoa,b,*, Kazumi Taguchia, Yasushi Ginozaa, Tomoyoshi Naganumaa, Yoshitomo Tanakaa, Katsuo Koikeb, Keizo Takatokua a

Research Center, Daiichi Radioisotope Laboratories, LTD., 453–1, Shimo-Okura, Matsuo-Machi, Sanbu-Gun, Chiba 289-1592, Japan Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Miyama 2–2-1, Funabashi, Chiba 274-8510, Japan

b

Received 1 July 2000; received in revised form 27 November 2000; accepted 2 December 2000

Abstract To diagnose and investigate neurodegenerative diseases affecting cholinergic neuron density, piperazine derivatives of vesamicol were synthesized and evaluated. Previously, we reported that trans-5-iodo-2-hydroxy-3-[4-phenylpiperazinyl] tetralin (DRC140, 1) possessed high selectivity for vesicular acetylcholine transporter (VAChT). In present study of the effect of alkyl substituents, we observed that the introduction of a methyl group into the ortho or meta positions of the phenyl group of 1 increased affinity for VAChT. trans-5-Iodo-2hydroxy-3-[4-[2-methylphenyl] piperazinyl]tetralin (2) displayed high affinity and specificity for VAChT. The regional distributions of radioactivity in the rat brain correlated well with known patterns of central cholinergic innervation. [123I]2 is a potentially useful compound for SPECT imaging. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Autoradiography; Cholinergic; Radioiodinated ligand; Synaptic vesicle; Vesamicol; Vesicular acetylcholine transporter

1. Introduction Cholinergic neurons projecting from the basal forebrain to the cerebral cortex and hippocampus are thought to be involved in cognitive function and memory. Because Alzheimer’s disease (AD) is a progressive disorder with dramatic loss of these cholinergic neurons, considerable attention has been paid to this aspect of the disease pathology; in addition, the cholinergic lesion has become a therapeutic target in AD. Data from postmortem samples of brain of AD patients suggest that deficiencies of choline acetyltransferase (ChAT), high-affinity choline transporter and acetylcholine esterase are initial neurochemical changes [4,21, 22,27]. Cholinergic neurotransmission requires the synthesis of acetylcholine in the cytoplasm, accumulation of acetylcholine in synaptic vesicles, and release of acetylcholine from the terminal of the cholinergic neuron. The vesicular acetylcholine transporter (VAChT) is responsible for the accumulation of acetylcholine into synaptic vesicles in the cho* Corresponding author. Tel.: ⫹81-479-86-4722; fax: ⫹81-479-863522. E-mail address: [email protected] (K. Bando).

linergic terminal. The VAChT is located exclusively in cholinergic neurons and has recently emerged as a useful marker for identifying the cholinergic terminal [1,11,24,26]. Therefore, specific ligands binding to VAChT are expected to provide a tool for studying the density or function of cholinergic neurons. The compound 2-(4-phenylpiperidino)-cycrohexanol (vesamicol) binds to the vesamicol binding site on VAChT protein with moderate affinity. There are many reports that have focused on vesamicol derivatives for single photon emission computed tomography (SPECT) and positron emission tomography (PET) [5,6,9,12,15,19,20]. In the human temporal cortex post-mortem, in vitro m-[125I]iodobenzyltrozamicol ([125I]MIBT) binding was reduced by 45% relative to age-matched controls [8]. SPECT studies showed decreased accumulation of [123I]iodobenzovesamicol ([123I]IBVM) in several brain areas in AD patients [16]. These compounds have a piperidine ring as a common structure. On the other hand, we have recently described trans - 5 - iodo - 2 - hydroxy - 3 - [4 - phenylpiperazinyl]tetralin (DRC140, 1) which replaced the piperidine ring of (-)iodobenzovesamicol with piperazine ring [2]. We have reported that this compound was a specific ligand to detect the VAChT. In binding studies using subcellular fraction of rat

0969-8051/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 0 ) 0 0 1 9 0 - 1

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brain, the highest [125I]1 binding was observed in synaptic vesicle fractions. We also observed that (-)-1 was not affected by ligands for other types of receptors, enzymes and transporters in inhibition studies. A particular advantage of the piperazine derivative is its high selectivity for VAChT over ␴ receptors. In vivo rat studies, the regional brain distribution of the piperazine derivative was consistent with the distribution of acetylcholine neuron. Rogers et al. [23] reported on some piperazine analogues testing for binding of Torpedo cholinergic synaptic vesicles to VAChT. They suggested that replacement of the piperidine ring of vesamicol by the piperazine ring lowers affinity for VAChT. However, replacement of the phenyl group of the resulting piperazine-containing compound with an otolyl group increases affinity for VAChT. The possibility that placing a methyl group in the phenyl ring of piperazine derivatives of benzovesamicol may increase potency is suggested by this fact. To synthesize SPECT agents using piperazine derivatives which have high affinity and specificity for VAChT and suitable biodistribution, the key point in drug design of the piperazine derivatives is to clarify the structure-activity relationship of the substituents of the phenyl ring. The present study was undertaken to determine the effect of alkyl substituents in phenyl ring of piperazine derivatives and biodistribution studies.

2. Materials and methods Most of the chemical reagents and solvents used to synthesize vesamicol analogs were purchased from Aldrich Chemical Company (Milwaukee, WI, USA) and Tokyo Chemical Industry (Tokyo, Japan). Proton nuclear magnetic resonance (NMR) spectra was recorded in JEOL JNMA500 (Tokyo, Japan). Chemical shifts were reported in parts per million (␦) relative to tetramethylsilane. Chemical shifts were expressed as ppm using CDCl3 as the internal standard. Coupling constants are given in hertz (Hz). NMR peak patterns were described by the following abbreviations: br ⫽ broad, s ⫽ singlet, d ⫽ doublet, t ⫽ triplet, m ⫽ multiplet. Fast atom bombardment mass spectra (FAB-MS) were obtained on JEOL JMS-HX100 spectrometer (Tokyo, Japan). High performance liquid chromatography (HPLC) were performed using a JASCO system consisting of PU980 pump, and equipped with a UV-detector (UV975) in series with a NaI (Tl) scintillation detector for radioactivity detection. Thin-layer chromatography (TLC) was performed on precoated MERCK Kieselgel 60 F254 (Darmstadt, Germany). Radio-TLC of radioactive synthesis was measured on Radiochromanizer (Aloka, Japan). Vesamicol, haloperidol and (⫹)SKF10,047 were purchased from Research Biochemicals International (Natick, MA, USA). [3H](⫹)-Pentazocine and [3H]1,3-di-o-tolyguanidine ([3H]DTG) were purchased from New England Nuclear (Boston, MA, USA). Male Wistar rats and Hartley

guinea pigs obtained from Japan SLC, LTD (Shizuoka, Japan) were housed in groups of five to six and two animals in a cage, respectively, with free access to food and water and maintained on a 12 hour light/dark cycle. All rats weighed between 170 –250 g at the time of preparation of tissue homogenates and biodistribution studies. All guinea pigs weighed between 300 –500 g at the time of preparation of tissue homogenate. 2.1. Chemical and radiochemical synthesis 2.1.1. (⫾)-trans-5-IODO-2-HYDROXY-3- [4-[2METHYLPHENYL] PIPERAZINYL] TETRALIN (2) 1-(2-Methylphenyl)piperazine hydrochloride (3.3 g, 15.2 mmol ) was dissolved in ethanol (40 mL) containing triethyl amine (2.2 mL) and then added to N-(trifluoroacetyl)-1amino-5,8-dihydronaphthalene oxide (2.0 g, 7.78 mmol). The mixture was heated to refluxing temperature for 20 hr. The solvent was evaporated and the crystalline residue was dissolved in methanol (14 mL) and treated with 2 N NaOH (40 mL). The mixture was stirred at room temperature for 20 hr. The reaction mixture was extracted with CH2Cl2. The organic layer was evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel with CH2Cl2/MeOH (98:2). The solvent was removed under high vacuum to provide the white solid. The solid was dissolved in acetic acid (2 mL) and concentrated HCl (1 mL) below 7°C. To the mixture was slowly dropped a solution of NaNO2 (262 mg) in water (15 mL) below 7°C and stirred for 20 min below 5°C. To the reaction mixture was slowly dropped a solution of KI (74 mg) and I2 (57 mg) in water (1 mL) below 7°C. The resulting mixture was stirred for 3.5 hr below 5°C and kept overnight at room temperature. With external cooling, the reaction mixture was neutralized by addition of 6 N NaOH and extracted with CH2Cl2. The organic extracts were combined, dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel with CH2Cl2/methanol (99:1). The solvent was removed under high vacuum to provide 2. The yield was 1.4 g (40%). The optical resolution of racemic 2 was performed by the same method described in literatures using (-)-␣-methoxy-␣-trifluoromethylphenylacethyl chloride [2,13]. 1H NMR(CDCl3)␦ 2.35 (s, 3H), 2.68 – 2.77 (m, 3H), 2.80 –2.89 (m, 2H), 2.97–3.06 (m, 7H), 3.25 (dd, J ⫽ 15.9, 5.5 Hz, 1H), 3.85 (ddd, J ⫽ 16.2, 10.4, 5.8 Hz, 1H), 4.33 (br, 1H), 6.84 (t, J ⫽ 7.6Hz, 1H), 6.98 –7.20 (m, 5H), 7.71 (d, J ⫽ 7.9Hz, 1H). FAB-MS m/z; 449 (MH⫹). 2.1.2. (⫾)-trans-5-IODO-2-HYDROXY-3- [4-[2, 5DIMETHYLPHENYL]PIPERAZINYL] TETRALIN (3) This compound was prepared as described for 2 using 1-(2,5-dimethylphenyl)piperazine (740mg, 3.89 mmol). The yield was 461 mg (26%). 1H NMR(CDCl3)␦ 2.30 (s, 3H), 2.31 (s, 3H), 2.67–2.76 (m, 3H), 2.80 –2.89 (m, 2H), 2.96 – 3.06 (m, 7H), 3.25 (dd, J ⫽ 16.2, 5.8 Hz, 1H), 3.85 (ddd, J ⫽ 16.2, 10.4, 5.8 Hz, 1H), 4.23(br, 1H), 6.81– 6.86 (m,

K. Bando et al. / Nuclear Medicine and Biology 28 (2001) 251–260

3H), 7.07–7.11 (m, 2H), 7.71 (d, J ⫽ 7.9Hz, 1H). FAB-MS m/z; 463(MH⫹). 2.1.3. (⫾)-trans-5-IODO-2-HYDROXY-3- [4-[2ETHYLPHENYL] PIPERAZINYL] TETRALIN (4) 1-[2-Ethylphenyl] piperazine was synthesized according to the method of Martin et al. [17]. Compound 4 was prepared as described for 2 using 1-[2-ethylphenyl] piperazine (941, 4.95 mmol). The yield was 483 mg (26%). 1H NMR(CDCl3)␦ 1.30 (t, J ⫽ 7.6Hz, 3H), 2.69 –2.80 (m, 5H), 2.82–2.90 (m, 2H), 2.97–3.08(m, 7H), 3.27 (dd, J ⫽ 15.9, 5.5 Hz, 1H), 3.87 (ddd, J ⫽ 16.2, 10.4, 5.8Hz, 1H), 4.17 (br,1H), 6.86 (t, J ⫽ 7.9Hz, 1H), 7.04 –7.14 (m, 4H), 7.18 – 7.21 (m, 1H), 7.73 (d, J ⫽ 7.9 Hz, 1H). FAB-MS m/z; 463(MH⫹). 2.1.4. (⫾)-trans-5-IODO-2-HYDROXY-3- [4-[2,3DIMETHYLPHENYL] PIPERAZINYL] TETRALIN (5) 1-[2,3-dimethylphenyl] piperazine was synthesized according to the method of Martin et al. [17]. Compound 5 was prepared as described for 2 using 1-[2,3-dimethylphenyl] piperazine (1.5 g, 7.88 mmol). The yield was 668 mg (19%). 1H NMR(CDCl3)␦ 2.27 (s, 3H), 2.28 (s, 3H), 2.68 – 2.78 (m, 3H), 2.81–2.89 (m, 2H), 2.96 –2.99 (m, 6H), 3.05 (dd, J ⫽ 16.2, 5.2 Hz, 1H), 3.25 (dd, J ⫽ 16.2, 5.8 Hz, 1H), 3.85 (ddd, J ⫽ 16.2, 10.4, 5.8 Hz, 1H), 4.22 (br, 1H), 6.84 (t, J ⫽ 7.6Hz, 1H), 6.91– 6.95 (m, 2H), 7.07–7.11 (m, 2H), 7.71 (d, J ⫽ 7.9Hz, 1H). FAB-MS m/z; 463 (MH⫹). 2.1.5. (⫾)-trans-5-IODO-2-HYDROXY-3- [4-[3METHYLPHENYL] PIPERAZINYL] TETRALIN (6) This compound was prepared as described for 2 using 1-[3-methylphenyl] piperazine (1.9 g, 7.79 mmol). The yield was 531 mg (30%).1H NMR(CDCl3)␦ 2.33 (s, 3H), 2.66 –2.71 (m, 1H), 2.75–2.79 (m, 2H), 2.81–2.88 (m, 2H), 2.98 –3.04 (m, 3H), 3.21–3.30 (m, 5H), 3.86 (ddd, J ⫽ 15.9, 10.4, 5.5 Hz, 1H), 4.12 (br, 1H), 6.71– 6.85 (m, 4H), 7.09 – 7.18 (m, 2H), 7.70 (d, J ⫽ 7.6Hz, 1H). FAB-MS m/z; 449 (MH⫹). 2.1.6. (⫾)-trans-IODO-2-HYDROXY-3- [4-[4METHYLPHENYL] PIPERAZINYL] TETRALIN (7) Compound 7 was prepared as described for 2 using 1-[4-methylphenyl] piperazine (1.9, 7.79 mmol). The yield was 589 mg (34%). 1H NMR(CDCl3)␦ 2.28 (s, 3H), 2.66 – 2.88 (m, 5H), 2.98 –3.04 (m, 3H), 3.17–3.27 (m, 5H), 3.86 (ddd, J ⫽ 16.2, 10.1, 5.8 Hz, 1H), 4.12 (br, 1H), 6.83 (t, J ⫽ 7.9Hz, 1H), 6.86 – 6.89 (m, 2H), 7.08 –7.11 (m, 3H), 7.70 (d, J ⫽ 7.9Hz, 1H). FAB-MS m/z; 449 (MH⫹). 2.1.7. (⫾)-trans-IODO-2-HYDROXY-3- [4-[2,6DIMETHYLPHENYL] PIPERAZINYL] TETRALIN (8) 1-[2,6-Dimethylphenyl] piperazine was synthesized according to the method of Martin et al. [17]. Compound 8 was prepared as described for 2 using 1-[2,6-dimethylphenyl] piperazine (2.13, 11.2 mmol) . The yield was 743 mg

253

(28%). 1H NMR(CDCl3)␦ 2.37 (s, 6H), 2.65–2.73 (m, 3H), 2.78 –2.94 (m, 4H), 3.04 (dd, J ⫽ 16.2, 4.9 Hz, 1H), 3.14 – 3.22 (m, 4H), 3.25 (dd, J ⫽ 16.2, 5.8Hz, 1H), 3.85 (ddd, J ⫽ 16.2, 10.4, 5.8 Hz, 1H), 4.26 (br, 1H), 6.84 (t, J ⫽ 7.6 Hz, 1H), 6.95–7.01 (m, 3H), 7.10 (d, J ⫽ 7.3Hz ,1H), 7.71 (d, J ⫽ 7.6Hz, 1H). FAB-MS m/z; 463 (MH⫹). 2.2. Radiolabeling and purification of [123I]2 and [123I]3 Radiolabeling and purification of [125I]1 was prepared using a method previously reported in the literature [2]. [125I]2 and [125I]3 were prepared by oxidative radioiododestannylation of tri-n-butyltin precursors. To a reaction vessel containing 50 ␮L of solution (20 ␮g/ml) of tributylstannyl precursor of 2 or 3 in ethanol was added 20 ␮L phosphate buffer (0.4 M, pH 1.3), 20 ␮L [125I]iodide in 0.1 N NaOH, and added consecutively 20 ␮L chloramine T (10 mg/mL) in water. After the mixture was kept at room temperature for 1 min, the reaction was quenched with 20␮L of an aqueous sodium of metabisulfate (100 mg/mL). Finally, one mL of a saturated sodium bicarbonate was added to the reaction vial. The resultant mixture was passed through a Sep-Pak C18 light column to trap the radioligand. After washing the SepPak with 8 mL water, the labeled ligand was eluted with 5 mL methanol. The eluent was then evaporated under reduced pressure. The residue was purified by HPLC (Shiseido AG 120, Tokyo Japan; methanol/ water/triethylamine ⫽ 80 : 20 : 0.2 as the mobile phase; flow rate 3 mL/min). [125I]2 or [125I]3 was obtained by evaporation under a nitrogen stream. The residue was dissolved in ethanol and the radioactivity measured. Chemical identity was checked after labeling by co-injection on HPLC of 2 or 3 in cold and radioactive forms under the same condition. 2.3. Sample preparations and binding assays for VAChT Adult male Wistar rats were sacrificed by decapitation and their brains were quickly removed from the skull. The brains without cerebellum were homogenized in a glassTeflon homogenizer using 12 strokes at 1000 rpm in ice cold buffered 0.32 M sucrose, containing 4 mM HEPESNaOH buffer, pH 7.4. The homogenate was centrifuged at 1100 g for 10 min. The resulting pellet was discarded, and the supernatant was collected and centrifuged at 9,200 g for 15 min. The supernatant was removed and the pellets were resuspended in assay buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, pH 7.4). The aliquots were stored at ⫺80°C until use. The competition assays were performed as follow. The fractions were incubated with 0.2 nM [125I]1 and various concentrations of competing compounds at 37°C for 3 hr in assay buffer. Nonspecific binding was defined using 50 ␮M (-)-vesamicol. The incubation was terminated by rapid filtration through Whatman GF/B filters previously soaked in assay buffer using a Brandel Cell Harvester. The filters were

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washed three times with 2 mL of ice-cold Tris-HCl buffer and counted using Minaxi gamma counter (Packard). Protein concentrations were determined with a Protein Assay kit (Bio-Rad) using a bovine serum albumin as a standard 2.4. Sample preparations and binding assays for ␴ receptors Guinea pigs and rats were sacrificed by decapitation. The guinea pig brain or the rat liver was quickly removed. Crude P2 membrane fraction was prepared from the brain, without cerebellum, of guinea pig or the liver of rat using the published method [18]. The tissues were homogenized in a glass-Teflon homogenizer using 10 strokes at 1000 rpm in ice cold buffered 0.32 M sucrose in 10 mM Tris-HCl, pH 7.4 (Trissucrose buffer) in a volume of 10 mg/g wet tissue weight. The homogenates were centrifuged at 4°C at 1000 g for 10 min and supernatants saved. The pellets were resuspended in 2 mL/g Tris-sucrose buffer and centrifuged at 4°C at 1000 g for 10 min. The supernatants from both 1000 g spins were combined and centrifuged at 4°C at 31,000 g for 15 min. The pellets were resuspended in 10 mM Tris-HCl, pH 7.4 in a volume of 3 mL/g of tissue and the suspensions were incubated for 15 min at 25°C. Following centrifugation at 31,000 g for 15 min, the pellets were resuspended in 10 mM Tris-HCl, pH 7.4. The aliquots were stored at ⫺80°C until use. The affinity of vesamicol derivatives for ␴-1 receptors was determined using ␴-1 selective ligand [3H](⫹)-pentazocine and brain P2 membranes of guinea pig. Briefly, the membranes (250 ␮g protein) were incubated with 3 nM [3H](⫹)-pentazocine and various concentrations (0.05–10000 nM) of vesamicol derivatives. Incubation was carried out in a total volume of 0.5 mL of 50 mM Tris-HCl, pH 8.0 for 120 min at room temperature. Non-specific binding was determined in the presence of 10 ␮M haloperidol. The affinity of vesamicol derivatives for ␴-2 binding assay was determined using rat liver P2 membranes (250 ␮g protein) and [3H]1,3-di-o-tolyguanidine ([3H]DTG). These were incubated with 3 nM [3H]DTG in the presence of 200 nM (⫹)-SKF10,047 to mask ␴-1 sites. Non specific binding was evaluated in the presence of 10 ␮M haloperidol. Assays were terminated by dilution with 2 ml ice-cold 10 mM Tris-HCl, pH 8.0 and vacuum filtration through Whatman GF/B filter glass fiber filters previously soaked in 0.5% polyethyleneimine using a Cell Harvester system (Inotech, Switzerland). The filters were then washed twice with 1 mL of ice-cold buffer. For measurement with MicroBeta (EG&G Wallac, Finland), the filters were dried and meltable solid scintillator, MeltiLex B/HS, was melted into the filters. Protein concentrations were determined with Protein Assay kit (BioRad) using a bovine serum albumin as a standard. 2.5. Data analysis Inhibitory concentration at 50% (IC50) value was determined by nonlinear curve fitting method, using EXCEL (Microsoft) incorporated Solver program.

2.6. In vivo binding studies Rats received an intravenous injection via tail vein with 100 ␮Ci of either [123I]2 or [123I]3 in 200 ␮L. After the various specified intervals after injection, the animals were sacrificed by decapitation under anesthesia with diethyl ether and blood sample was collected into heparinized test tube. The brain was removed and immediately placed on ice, and dissected into segments consisting of the frontal cortex, occipital cortex, hippocampus, striatum, cerebellum and other regions. The peripheral tissue samples were excised from each animal. The tissue was weighted and the radioactivity of each tissue was measured using a Minaxi gamma counter (Packard). The %dose/gram of tissue was determined by comparison of the tissue counts to suitable diluted aliquots of each injected radiotracer. In order to study specific binding of [125I]2 and [125I]3 in vivo using autoradiographic analysis, a rat was intravenously injected with 80 ␮Ci of either [125I]2 or [125I]3 in 400 ␮L and aminobenzovesamicol [0.2␮mole/kg for nonspecific binding] at 5 min prior to the radioligand injection. The rat was sacrificed by decapitation at 240 min post i.v. injection of the radiotracer. The brain was removed and immediately frozen. Coronal sections were cut by a cryostat at ⫺20°C. The sections were thaw-mounted onto slide, air-dried and placed on an imaging plate in X-ray cassettes. After exposure the plates were scanned using a Fuji Bioimaging Analysis System BAS-1800 (Fuji film, Tokyo, Japan).

3. Results and discussion 3.1. Radiolabeling and purification of (-)-2 and (-)-3 The preparations of [125I]2 and [123I]2 was based on destannylation of the corresponding tributylstannyl precurser by the chloramine-T method (Fig. 1). For labeling (-)-2, the radioiodinated products were obtained in greater than 50% yield for [125I] and [123I] labeling. The compound was purified by HPLC using a reversed-phase C18 column. After purification, each of the radiochemical purities was found on average to be greater than 99%. As these compounds were obtained without a carrier, they displayed a high specific activity of ⬎2,000 and ⬎50,000Ci/mmol for [125I]2 and [123I]2, respectively. Chemical identity was checked after labeling by co-injection on HPLC of 2 in cold and radioactive forms. [125I]3 and [123I]3 were prepared as described for [125I]2 and [125I]2. The radioiodinated products were obtained in greater than 50% yield for [125I] and [123I] labeling. After purification by HPLC, the radiochemical purity was greater than 99%. They displayed a high specific activity of ⬎2,000 Ci/mmol and ⬎50,000 Ci/mmol for [125I]3 and [123I]3, respectively.

K. Bando et al. / Nuclear Medicine and Biology 28 (2001) 251–260

Fig. 1. Synthesis and radiolabeling with

123

I or

255

125

I of piperazine derivatives of vesamicol.

Table 1 Potencies of various compounds to inhibit the binding of [125I]1, [3H](⫹)- pentazocine and [3H]DTG Compound

1 2 3 4 5 6 7 8

R1

R2

H CH3 CH3 CH2CH3 H H H CH3

H H H H H H CH3 H

R3

H H CH3 H CH3 CH3 H H

(⫺)-1* (⫹)-1* (⫺)-2 (⫹)-2 (⫺)-3 (⫹)-3 Compound

1 2 3 4 5 6 7 8 (⫺)-1* (⫹)-1*

sigma-1 [3H]-(⫹)-pentazocine

R4

H H H H CH3 H H H3

Vesamicol [125I]1 IC50(nM)

nH

4.14 ⫾ 0.31 0.74 ⫾ 0.02 1.16 ⫾ 0.11 1.10 ⫾ 0.02 1.67 ⫾ 0.25 2.47 ⫾ 0.1 14.74 ⫾ 1.55 6.94 ⫾ 0.4

1.06 ⫾ 0.07 0.96 ⫾ 0.06 0.97 ⫾ 0.09 0.82 ⫾ 0.03 0.93 ⫾ 0.04 0.87 ⫾ 0.04 1.05 ⫾ 0.13 1.00 ⫾ 0.04

1.52 ⫾ 0.12 70.30 ⫾ 13.2 0.51 ⫾ 0.05 1.964 ⫾ 0.08 0.83 ⫾ 0.09 11.45 ⫾ 1.23

0.99 ⫾ 0.04 0.84 ⫾ 0.05 1.05 ⫾ 0.07 0.97 ⫾ 0.07 1.02 ⫾ 0.07 0.97 ⫾ 0.07

sigma-2 [3H]-DTG

IC50(nM)

nH

IC50(nM)

nH

4481 ⫾ 183 ⬎5000 ⬎5000 4563 ⫾ 288 ⬎5000 4776 ⫾ 254 ⬎5000

1.04 ⫾ 0.05

⬎5000 ⬎5000 4854 ⫾ 92.3 2968 ⫾ 137 ⬎5000 3126 ⫾ 123 ⬎5000

0.93 ⫾ 0.08

3371 ⫾ 210 3336 ⫾ 340

1.01 ⫾ 0.05 1.00 ⫾ 0.03 1.03 ⫾ 0.02 1.06 ⫾ 0.1

The data are the mean ⫾ S.D. of three experiments with duplicate determinations. * Reproduced from reference 2.

2120 ⫾ 145 1940 ⫾ 92.5

1.03 ⫾ 0.10 1.28 ⫾ 0.04 0.84 ⫾ 0.05 0.87 ⫾ 0.02 0.99 ⫾ 0.04

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Fig. 2. Biodistribution in rats after an injection of [123I]2 (A) and [123I]3 (B). Data are presented as means of normalized injected dose per gram of tissue (%dose/g) ⫾ S.D; n⫽4 for each tracer and time point.

3.2. In vitro binding studies Vesamicol possesses affinities for ␴-1 and ␴-2 receptors [3,7], vesamicol binding protein in noncholinergic neuron [10] and ␣-adrenergic blocking activity [25]. In previous studies, we described that [125I]1 binding using rat brain homogenate showed high selectivity for the VAChT [2]. For this reason, we utilized this tracer for assessment of the potency of synthesized piperazine derivatives for VAChT. The potency of piperazine derivatives for VAChT and ␴-1 and ␴-2 receptors are showed in Table 1. All derivatives of vesamicol inhibited [125I]1 binding in rat brain homogenate. Comparison of the inhibitory activity for VAChT showed that affinities vary according to the alkyl position of phenyl ring in the derivatives. The IC50 (0.74 nM) of 2 was 5-fold more potent than that of 1. A methyl group substituents in ortho position of phenyl ring increased potency for VAChT. These observations are consistent with the results

for structure-activity relationship of vesamicol derivative [23]. The potency of 7 to which is added methyl group at para-position of phenyl ring extremely reduced activity for VAChT (IC50 ⫽ 14.7 nM). The potency of ethyl derivatives at ortho position, 4, also increase approximately four-fold, compared with that of 1. However, addition of two methyl groups at both para positions of phenyl ring in 1 structure yielded a strong reduction in VAChT potency (IC50 ⫽ 6.94 nM for 8). It may be that the dihedral angle and rotation between phenyl ring and piperazyl ring alters the suitable conformation by addition of methyl at ortho the position of one side. The meta position derivative, 6, was only modestly (1.7-fold) more potent than 1. Unexpectedly, 3 and 5 to which are added two methyl group at ortho and para position of phenyl ring did not increase potency for the receptors, as compared to 2. Some derivatives of vesamicol have been reported to possess affinity for ␴ receptors [3,7,13]. To determine

K. Bando et al. / Nuclear Medicine and Biology 28 (2001) 251–260

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Fig. 3. Time-activity curves of [123I]2 (A) and [123I]3 (C) of rat brain after intravenous injection of the tracers. Data are presented as means of normalized injected dose per gram of tissue (%dose/g) ⫾ S.D; n⫽4 for each tracer and time point. Regions-to-cerebellum ratios of [123I]2 (B) and [123I]3 (D). Data are presented as means of each region/cerebellum radioactivity ratio ⫾ S.D; n⫽4.

whether the piperazine derivatives have potency toward ␴ receptors, we performed competition studies with [3H](⫹)pentazocine and [3H]DTG in the presence of 200 nM (⫹)SKF10,047 as ␴-1 and ␴-2 receptor ligands, respectively. All derivatives tested showed extremely weak affinity for ␴-1 and ␴-2 receptors (IC50 ⬎ 1000 nM). Consequently, an advantage of the piperazine derivatives is its high selectivity for VAChT over ␴ receptors. 3.3. In vivo biodistribution We conducted biodistribution studies of two analogs (2 and 3) which have high potency and are close to unity with the Hill coefficient. The time-activity data in rat brain regions, blood (%dose/g tissue) and peripheral organs (%dose) after intravenous injection of [123I]2 or [123I]3 are shown in Fig. 2 and 3. High radioactivity was observed in small intestine with maximum accumulation 59%dose at 90 min and decreased. The radioactivity of large intestine rose dramatically at 240 min in agreement with the decrease of

activity in small intestine. It seems likely that the main excretion root [123I]1 is hepatobiliary excretion system. In order to evaluate in vivo deiodination of [123I]2 or [123I]3, we removed the whole thyroid gland of each animal and measured its radioactivity. This radioactivity remained constant at low level through each sampling time. The radioactivity of each ligand in the whole brain was gradually washed out. [123I]2 showed higher radioactivity in whole brain at 5 min after injection than [123I]3, 0.780 ⫾ 0.084%dose/g for [123I]2 and 0.601 ⫾ 0.040%dose/g for [123I]3. Our previous studies provided evidence that [123I]1 readily crosses the blood-brain barrier and displays a regional distribution [2]. [123I]1 showed the highest brain uptake at 5 min after injection in the three radioligands (1.618 ⫾ 0.272 dose/g for [123I]1). The structure of 2 or 3 differs in the presence of one or two methyl substituents of phenyl ring from that of 1, respectively. Although 2 or 3 were more lipophilic than 1, both radioligands show lower uptake in brain. Kessler et al. reported an inverted parabolic relationship between striatum uptake of [125I]labeled benz-

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Fig. 4. Autoradiographic distribution of [125I]2 (A and B) and [125I]3 (C and D) rat brain at 240 min after injection in coronal section. Effect of aminobenzovesamicol [0.2 ␮mole/kg, i.v.] of preinjection at 5 min prior to the radioligand injection on the distribution of the radioactivity (B and D). ME, medial eminence; VCA, anterior ventral cochlear nucleus; Tu, olfactory tubercle; IPA, interpeduncular nucleus; BLA, basolateral amygdaloid; 7n, facial nerve; CPu, striatum; Rt, reticular thalamus; Hpc, hippocampus; CTX, cerebral cortex, Cer, cerebellum.

amides as dopamine D2 receptor ligand and lipophilicity [14]. According to their presumed explanation for the relationship of cerebral uptake of the tracer and lipophilicity, low lipophilic ligands do not cross the blood-brain barrier efficiently whereas high lipophilic molecules strongly bind to plasma proteins and cell membranes. For this reason, a small fraction of higher lipophilic 3 in plasma may be able to freely cross the blood-brain barrier. The radioactivity of [123I]2 in cerebellum showed lower uptake than that of [123I]3. Using cerebellum concentration for the estimation of nonspecific binding, we found that [123I]3 was higher in nonspecific binding and slower in clearance than [123I]2 (biological half life t1/2 ⫽ 3.4 hr for [123I]2 and t1/2 ⫽ 11.2 hr for [123I]3). This is in agreement with higher lipophilicity of 3 than that of 2 (calculated log P ⫽ 4.07 for [123I]2 and log P ⫽ 4.49 for [123I]3, using Pallas program ver.1.2 by CompuDrug Chemistry Ltd., CA, USA). High lipophilicity causes slow clearance from nonspecific binding into the brain. The distribution in regional brain of [123I]2 and [123I]3

are shown in Fig 3. A gradual decline in radioactivity was observed in all brain areas, except the striatum, with both radioligands. On the other hand, a high level of accumulation in the striatum remained at almost the same level until 240 minutes. The distribution of [123I]2 in each region of rat brain at 240 min after injection showed the following order: striatum (%dose/g ⫽ 0.581 ⫾ 0.038), frontal cortex (%dose/ g ⫽ 0.164 ⫾ 0.010), hippocampus (%dose/g ⫽ 0.123 ⫾ 0.0073), occipital cortex (%dose/g ⫽ 0.212 ⫾ 0.020), and cerebellum (%dose/g ⫽ 0.038 ⫾ 0.003). In later periods after injection of each radiotracer, the regional distribution pattern of radioactivity in the brain correlated well with the cholinergic innervation though the rat brain. Furthermore, the regional distribution pattern in brain of both tracers, [123I]2 and [123I]3, in rat was quite similar to that of radioligand of VAChT for SPECT or PET previously described in the literature [2,5,12,13]. Tissue-to-cerebellum ratios increased linearly over the observation period for [123I]2. The ratios reached values of 15.4 ⫾ 0.69 in striatum, 4.3 ⫾ 0.31 in frontal cortex, 5.6 ⫾

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0.37 in occipital cortex and 3.3 ⫾ 0.09 in hippocampus at 240 minutes postinjection. Although the ratios for [123I]2 were higher than those for [123I]3, a similar linear increase of the ratios was observed in regional distribution of [123I]3. (10.4 ⫾ 1.78 in striatum, 3.80 ⫾ 0.43 in frontal cortex, 3.2 ⫾ 0.41 in occipital cortex and 2.5 ⫾ 0.32 in hippocampus at 240 min postinjection). A rate of cerebellar washout of [123I]3 was slow when compared with that of [123I]2. 3.4. Autoradiography Ex vivo autoradiographic localization of [125I]2 and [ I]3 binding sites in rat brain 240 min after injection of radioligands are sown in Figure 4. The striatum displayed the highest levels of binding of both ligands. High levels of [125I]2 and [125I]3 also were found in striatum, interpeduncular nucleus, basolateral amygdala and olfactory tubercle, medial eminence and moderate accumulations were found in cortex and hippocampus, reticular thalamic nucleus, anterior ventral cochlear nucleus and facial nucleus. These data are in agreement with the areas that are well-known as high density regions of acetylcholine transporters [1,11,24]. In rats pretreated with aminobenzovesamicol, the resultant density in cortex and hippocampus of the corresponding matched sections at 240 min postinjection were reduced to almost background level, like that in the cerebellum. 125

4. Conclusion We have developed a series of piperazine derivatives that have a high specificity for VAChT. To develop new ligand for SPECT agents, the inhibition studies demonstrated that the advantage of this structure, piperazine derivative, is its high selectivity for VAChT over ␴ receptor. Two compounds of radioiodinated piperazine derivatives, [123I]2 and [123I]3, have been evaluated in rat. The biodistribution of both ligands seem to represent high-density vesicular acetylcholine transporter regions. However, [123I]3 was not a useful ligand as a SPECT tracer because of slow clearance from nonspecific region. [123I]2 may be a more viable compound for SPECT agent.

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