474
Vol. 54, No. 4
522.1010.
man GF/B glass fiber filters presoaked in 0.5% polyethylenimine for at least
1-(3,4-Dimethoxyphenylacetyl)-4-[3-(3-iodophenyl)propionyl]piperazine 30 min at room temperature prior to use, followed by a rapid washing 3
(3b): Yield 90.5%, IR (CHCl3): 2962, 1639, 1515, 1459, 1261 cmꢄ1
NMR (CDCl3): 2.59 (2H, t, Jꢂ7.5 Hz, phCH2CH2), 2.90 (2H, t, Jꢂ7.5 Hz,
.
1H-
times with 4 ml of ice-cold 10 mM Tris–HCl buffer (pH 8.0). The radioactiv-
ity trapped on the filters was counted by scintillation spectrometer after an
phCH2CH2), 3.29—3.58 (8H, m, piperazine), 3.68 (2H, s, phCH2CO), 3.86 overnight extraction of counts in 10 ml scintillation fluid. These IC50 values
(6H, s, OCH3ꢃ2), 6.76—6.83 (3H, m, aromatics), 6.99—7.55 (4H, m, aro- were determined from displacement curves of the percent inhibition of
matics). CI-HR-MS Calcd for C23H27IN2O4 (MHꢀ) m/z: 522.1017. Found: [3H] DTG binding.
522.1014. Sigma-1 and sigma-2 receptor binding assays were examined the same
1-(3,4-Dimethoxyphenylacetyl)-4-[3-(4-iodophenyl)propionyl]piperazine manner described above except using [3H]-(ꢀ)-pentazocine (0.1 nmol,
(3c): Yield 86.7%, IR (CHCl3): 2930, 1640, 1515, 1431, 1261 cmꢄ1
NMR (CDCl3): 2.59 (2H, t, Jꢂ7.8 Hz, phCH2CH2), 2.90 (2H, t, Jꢂ7.8 Hz,
phCH2CH2), 3.33—3.58 (8H, m, piperazine), 3.69 (2H, s, phCH2CO), 3.87
(6H, s, OCH3ꢃ2), 6.76—6.83 (3H, m, aromatics), 6.96 (2H, d, Jꢂ8.4 Hz,
.
1H-
0.46 kBq) and [3H] DTG (0.1 nmol, 0.46 kBq) in the presence of carbetapen-
tane (0.1 mM) to mask sigma-1 binding site, respectively.
Synthesis of Precursors. Substitute 1-[2-(3,4-Dimethoxyphenyl)-
ethyl]-4-[3-(tributylstannylphenyl)propyl]piperazine (5a—c) The mix-
aromatics), 7.60 (2H, d, Jꢂ8.4 Hz, aromatics). CI-HR-MS Calcd for ture of compound 4a—c (0.20 g, 0.40 mmol) and bistributyltin (0.64 g,
C23H27IN2O4 (MHꢀ) m/z: 522.1017. Found: 522.1007.
Substitute 1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piper- anhydrous toluene (7 ml) was refluxed under argon for 17 h. After cooling,
azine (4a—c) The compounds 3a—c (0.91 g, 2.28 mmol) were dissolved the reaction mixture was filtered through celite. The filtrate was concentrated
1.12 mmol) and tetrakis(triphenylphosphine)palladium (30 mg, 26 mmol) in
in anhydrous tetrahydrofuran (5 ml), followed by, 1.0 M diborane-tetrahydro-
in vacuo. The oily residue was purified by silica gel column chromatography
furan solution were dropwised (17.0 ml) at room temperature under argon (MeOH/CHCl3ꢂ1/20) affording 4a—c.
gas. The reaction mixture was heated at reflux for 18 h. The resulting solu-
tion was added 1 M HCl (25 ml) at 0 °C, and stirred for 15 min at room tem-
perature. After refluxed for 1 h, the solution was cooled, and evaporated in
vacuo to reduce tetrahydrofran. The residue was added 4 M NaOH until pH
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(2-tributylstannylphenyl)propyl]-
piperazine (5a): Yield 47.5%, IR (CHCl3): 3018, 2398, 1518, 1422,
1225 cmꢄ1 1H-NMR (CDCl3): 0.86—1.57 (27H, m, Bu3), 1.83 (2H, q,
Jꢂ7.5 Hz, phCH2CH2CH2), 2.41 (4H, t, Jꢂ7.5 Hz, CH2N), 2.56—2.64
.
11 and the mixture was extracted with CHCl3 (20 mlꢃ3). The organic layer (10H, m, phCH2CH2CH2 and piperazine), 2.76 (2H, t, Jꢂ7.5 Hz,
was dried over K2CO3 and the solvent was evaporated in vacuo to afford the phCH2CH2N), 3.87 (6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics),
crude product as yellow oil. This oil was purified by silica gel column chro- 7.10—7.35 (4H, m, aromatics). CI-HR-MS Calcd for C35H58N2O2Sn (MHꢀ)
matography (MeOH/CHCl3ꢂ1/20), the oil product was converted to its hy- m/z: 658.3520. Found: 658.3514.
drobromide salt. Recrystallized from methanol afforded 4a—c.
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(3-tributylstannylphenyl)propyl]-
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(2-iodophenyl)propyl]piperazine piperazine (5b): Yield 45.0%, IR (CHCl3): 3018, 2398, 1518, 1422,
1
(4a): Yield 80.8%, mp 246—248 °C, IR (KBr): 2910, 2439, 1521, 1462, 1225 cmꢄ1. H-NMR (CDCl3): 0.86—1.57 (27H, m, Bu3), 1.83 (2H, q,
1267 cmꢄ1
.
1H-NMR (Free Base, CDCl3): 1.80 (2H, q, Jꢂ7.8 Hz,
Jꢂ7.5 Hz, phCH2CH2CH2), 2.41 (4H, t, Jꢂ7.5 Hz, CH2N), 2.56—2.64
phCH2CH2CH2), 2.37 (4H, t, Jꢂ7.8 Hz, CH2N), 2.56—2.62 (10H, m, (10H, m, phCH2CH2CH2 and piperazine), 2.76 (2H, t, Jꢂ7.5 Hz,
phCH2CH2CH2 and piperazine), 2.77 (2H, t, Jꢂ7.8 Hz, phCH2CH2N), 3.87 phCH2CH2N), 3.87 (6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics),
(6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics), 6.98—7.56 (4H, m, aro-
7.10—7.35 (4H, m, aromatics). CI-HR-MS Calcd for C35H58N2O2Sn (MHꢀ)
matics). CI-HR-MS (Free Base) Calcd for C23H31IN2O2 (MHꢀ) m/z: m/z: 658.3520. Found: 658.3517.
494.1432. Found: 494.1423. Anal. Calcd for C23H31IN2O2·2HBr: C, 42.10;
H, 5.07; N, 4.27. Found: C, 41.93; H, 5.26; N, 4.24.
Radiolabeling Aqueous hydrogen peroxide (10 ml, 30%, w/v) was
added to a mixture of [125I] NaI (10 ml, 37.0 MBq, 74 TBq/mmol), 0.1 N HCl
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(3-iodophenyl)propyl]piperazine (25 ml) and tributylstannyl precursor 5a, b (0.01 mg in 10 ml ethanol) in a
(4b): Yield 92.6%, mp 247—249 °C, IR (KBr): 2910, 2439, 1521, 1462, sealed vial. After stirring for 10 min at room temperature, the reaction mix-
1267 cmꢄ1
.
1H-NMR (Free Base, CDCl3): 1.80 (2H, q, Jꢂ7.8 Hz,
ture was quenched with aqueous sodium bisulfite (0.1 mg in 10 ml). The
phCH2CH2CH2), 2.37 (4H, t, Jꢂ7.8 Hz, CH2N), 2.56—2.62 (10H, m, mixture was isolated by HPLC using NaH2PO4 solution (10 mM)/MeOH
phCH2CH2CH2 and piperazine), 2.77 (2H, t, Jꢂ7.8 Hz, phCH2CH2N), 3.87 (15/85) as an eluent at a flow rate of 3.0 ml/min. The fraction corresponding
(6H, s, OCH3ꢃ2), 6.73—6.81 (3H, m, aromatics), 6.98—7.56 (4H, m, aro-
of 4a, b was collected and the solvent was removed in vacuo. The retention
matics). CI-HR-MS (Free Base) Calcd for C23H31IN2O2 (MHꢀ) m/z: times were 13.8 min for [125I] 4a and 14.5 min for [125I] 4b. The radiochemi-
494.1432. Found: 494.1414. Anal. Calcd for C23H31IN2O2·2HBr: C, 42.10; cal yields based on [125I] NaI were 90.0 and 94.0%, respectively. The radio-
H, 5.07; N, 4.27. Found: C, 42.16; H, 5.04; N, 4.09.
1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-[3-(4-iodophenyl)propyl]piperazine than 99% and approximately 74 TBq/mmol.
(4c): Yield 91.4%, mp 247—249 °C. IR (KBr): 2938, 2434, 1517, 1451, In Vitro Binding Studies Crude P2 membrane fractions of rat brain
chemical purity and the specific activities of all three tracers were greater
1264 cmꢄ1
.
1H-NMR (Free Base, CDCl3): 1.75 (2H, q, Jꢂ7.8 Hz,
(2.0 mg of protein) in Tris–HCl (50 mM, pH 8.0) were incubated for 90 min
phCH2CH2CH2), 2.33 (4H, t, Jꢂ7.8 Hz, CH2N), 2.49—2.58 (10H, m, at 25 °C with [125I] 4a or [125I] 4b (1—300 nM, 0.74 kBq, respectively). The
phCH2CH2CH2 and piperazine), 2.71 (2H, t, Jꢂ7.8 Hz, phCH2CH2N), 3.80 total incubation volume was 1.0 ml. Nonspecific binding was determined in
(6H, s, OCH3ꢃ2), 6.67—6.74 (3H, m, aromatics), 6.87 (2H, d, Jꢂ8.4 Hz,
the presence of haloperidol (10 mM). Specific binding was defined as the dif-
aromatics), 7.52 (2H, d, Jꢂ8.4 Hz, aromatics). CI-HR-MS (free base) Calcd ference between the total and the nonspecific binding. Assays were termi-
for C23H31IN2O2 (MHꢀ) m/z: 494.1432. Found: 494.1424. Anal. Calcd for nated by rapid vacuum filtration through Whatman GF/B glass fiber filters
C23H31IN2O2·2HBr: C, 42.10; H, 5.07; N, 4.27. Found: C, 42.00; H, 5.00; N, presoaked in 0.5% polyethylenimine for at least 30 min at room temperature
4.05.
Preparation of Rat Brain Membranes Receptor binding assays were
prior to use, and assayed filters were washed 6 times with 4 ml ice-cold
10 mM Tris–HCl buffer (pH 7.4). The filter-bound radioactivity was counted
performed using the crude synaptosomal (P2) membrane fraction of rat in an auto-g-counter. The saturation binding data were analyzed by
brain, according to a modified the method of Leitner et al.44) Male Wistar Scatchard plot analysis for determination of the equilibrium dissociation
rats (250—300 g) were decapitated and the brain, minus cerebellum, were constant (Kd), and the maximal number of binding site (Bmax).
homogenized in 10 volumes of ice-cold 10 mM Tris–HCl/0.32 M sucrose
buffer (pH 7.4), using a Polytorone homogenizer at 24000 rpm for 20 s. The
In Vitro Blocking Studies Crude P2 membrane fractions of rat brain
(2.0 mg of protein) were incubated in Tris–HCl (50 mM, pH 8.0) for 90 min
homogenates were centrifuged at 1000 g for 10 min at 4 °C to remove cell at 25 °C with [125I] 4a or [125I] 4b (0.74 kBq) and various receptor ligand
debris. The supernatants were centrifuged at 45000 g for 15 min at 4 °C. The
final pellets were taken and adjusted to a protein concentration of 2 mg/ml in DTG, (ꢀ)-3-PPP, (ꢁ)-SKF10047, (ꢀ)-SCH23390, (ꢄ)-sulpiride, (ꢄ)-nico-
(1 mM, 100 ml), 4a—c, SA4503, haloperidol, rimcazole, carbetapentane,
ice-cold 10 mM Tris–HCl buffer (pH 7.4). Protein concentration was deter-
mined according to the Lowly method.46)
tine, phentolamine, atropine, and naloxone. The total incubation volume was
1.0 ml. Assays were terminated by rapid vacuum filtration through Whatman
In Vitro Competitive Binding Assays Sigma receptor binding assay GF/B glass fiber filters, presoaked in 0.5% polyethylenimine for at least
was carried out under the follow conditions. Crude P2 membrane fractions 30 min at room temperature prior to use. These filters were washed 6 times
of rat brain (2.0 mg of protein) were incubated with 0.1 nmol [3H] DTG
(0.46 kBq) and the test compound (3.3ꢃ10ꢄ9—1.0ꢃ10ꢄ5 M) in 50 mM
Tris–HCl (pH 8.0) at 25 °C for 90 min. The total incubation volume was
1.0 ml. Nonspecific binding were determined in the presence of haloperidol
(10 mM). Assays were terminated by rapid vacuum filtration through What-
with 4 ml ice-cold Tris–HCl buffer (10 mM, pH 7.4). The filter-bound ra-
dioactivity was counted in an auto-g-counter.
Acknowledgements This work was supported by Grant-in-Aid for Sci-
entific Research (C) No. 13670972, 17591298 and High Technology Re-