386
T. Masuko, T. Kusama, H. Nagaoka, K. Metori, Y. Kizawa, and M. Miyake
Vol 45
NMR was recorded on a JEOL JNM-GSX 400 spectrometer
containing tetramethylsilane as the standard. Mass spectra were
taken on a JEOL JMS-GCmate instrument. Adult female
Xenopus laevis were chilled on ice, and the preparation and
maintenance of oocytes performed as described previously [8,9].
Capped cRNAs were prepared from linearized cDNA templates
using mMessage mMachine kits (Ambion, Austin, TX). Oocytes
were injected with NR1A and NR2 cRNAs at a ratio of 1:5 (0.2-
4 ng of NR1A plus 1-20 ng of NR2). Macroscopic currents were
recorded with a two-electrode voltage-clamp using the Dual
Electrode Voltage Clamp Amplifier CEZ-1250 (Nihon Koden,
Tokyo, Japan). Electrodes were filled with 3M KCl. Oocytes
were continuously superfused (ca. 5 ml/min) with a Mg2+-free
saline solution (96 mM NaCl, 2 mM KCl, 1.8 mM BaCl2, 10
mM HEPES, pH 7.5). This solution contained BaCl2 rather than
Hz), 7.13—7.19 (6H, m), 7.23—7.26 (4H, m). HR-ms (FAB)
m/z: 517.3331 (Calcd for C35H41N4: 517.3331).
N,N'-(6H,12H-5,11-Methanodibenzo[b,f][1,5]diazocine-2,8-
diyldi-2,1-ethanediyl)-bis[N-(2-methoxycarbonylethyl)phen-
ethylamine] (4). A mixture of 3b (200 mg, 0.38 mmol), methyl
.
acrylate (196 mg, 2.28 mmol) and Cu(OAc)2 H2O (8 mg, 0.04
mmol) in MeOH (5 ml) was stirred for 24 h at 100 °C under N
2
atmosphere. The mixture was concentrated under reduced
pressure. The residue was purified by column chromatography
on silica gel with CHCl3:MeOH (10:1) and EtOAc:MeOH (9:1)
as the eluent to give 230 mg, 88% as a pale yellow oil, which
1
was used for the next reaction without further purification. H
nmr (CDCl3) δ:2.44 (4H, t, J =7.1 Hz), 2.59-2.72 (16H, m),
2.89 (4H, t, J =7.1 Hz), 3.64 (6H, s), 4.11 (2H, d, J =16.8 Hz),
4.29 (2H, s), 4.65 (2H, d, J =16.8 Hz), 6.69 (2H, d, J =1.7 Hz),
6.96 (2H, dd, J =8.0, 1.7 Hz), 7.04 (2H, d, J =8.0 Hz), 7.13—
7.20 (6H, m), 7.24—7.28 (4H, m). HR-ms (FAB) m/z: 689.4074
(Calcd for C43H53N4O4: 689.4066).
+
CaCl2, and, in most experiments, oocytes were injected with K -
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA; 100 nl of 40 mM solution at pH 7.5) on the day of
recording to eliminate Ca2+-activated Cl- currents [8,9].
Glutamate and glycine were purchased from Wako Pure
Chemical Industries, Ltd (Osaka, Japan). BAPTA were
purchased from Sigma (St. Louis, MO).
N,N'-(6H,12H-5,11-Methanodibenzo[b,f][1,5]diazocine-2,8-
diyldi-2,1-ethanediyl)-bis[N-(3-hydroxyxycarbonylethyl)-
phenethylamine] (5a). Compound 4 (170 mg) in THF (5 ml)
was added dropwise to a stirred suspension of LiAlH4 (57 mg,
1.5 mmol) in THF (5 ml) at room temperature. After 12 h, H2O
(57 ml), 15% NaOH (57 ml), and H2O (171 ml) was added
slowly in that order under stirring at room temperature, and then
K2CO3 was added and stirring was continued for 0.5 h at room
temperature. The mixture was filtered and the filtrate then
evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel with CHCl3:MeOH (10:1)
as the eluent to give 158 mg, 100% as a pale yellow oil, which
2,8-Bis(phenethylcarbonylmethyl)-6H,12H-5,11-methano-
dibenzo[b,f][1,5]-diazocine (3a). A mixture of 6H,12H-5,11-
methanodibenzo[b,f][1,5]diazocine-2,8-diylaceticacid pentaflu-
orophenyl ester 2 (670 mg, 1 mmol), 2-phenethylamine (242
mg, 2 mmol) and TEA (0.28 mL, 2 mmol) in CH2Cl2 (40 ml)
was stirred at room temperature. After 15 h, the reaction mixture
was diluted with CH2Cl2 (150 ml). The organic layer was washed
with H2O and dried over MgSO4, and evaporated under reduced
pressure to afford a white solid, which was chromatographed on
a silica gel column with CHCl3:MeOH (10:1) as the eluent to
give a white solid. This white solid was then washed with
EtOAc to give a white powder (0.52 g, 96%). An analytical
sample was obtained by recrystallizing this material from
1
was used for the next reaction without further purification. H
nmr (CDCl3) δ: 1.66—1.71 (6H, m), 2.64—2.76 (18H, m),
3.68—3.70 (2H, m), 3.86 (4H, t, J =5.6 Hz), 4.12 (2H, d, J =16.8
Hz), 4.29 (2H, s), 4.65 (2H, d, J =16.8 Hz), 6.70 (2H, d, J =1.4
Hz), 6.97 (2H, dd, J =8.0, 1.4 Hz), 7.05 (2H, d, J =8.0 Hz),
7.15—7.21 (6H, m), 7.26—7.29(4H, m). HR-MS (FAB) m/z:
633.4168 (Calcd for C41H53N4O2: 633.4168).
1
CHCl3-hexane, cottony colorless needles (mp 243—244 °C). H
nmr (CDCl3) δ:2.71 (4H, t, J =6.8 Hz), 3.38 (s, 4H), 3.42 (4H,
q, J =6.8 Hz), 4.11 (2H, d, J =16.8 Hz), 4.28 (2H, s), 4.64 (2H,
d, J =16.8 Hz), 5.35 (2H, br s), 6.72 (2H, s), 6.97 (2H, dd, J =6.8
Hz, 1.7 Hz), 7.02—7.04 (4H, m), 7.08 (2H, d, J =8.1 Hz),
7.19—7.23 (6H, m). HR-ms (FAB) m/z: 545.2916 (Calcd for
C35H37N4O2: 545.2916). Anal: Calcd for C35H36N4O2: C, 77.18;
H, 6.66; N, 10.29. Found: C, 76.98; H, 6.63; N, 10.23.
N,N'-(6H,12H-5,11-Methanodibenzo[b,f][1,5]diazocine-2,8-
diyldi-2,1-ethanediyl-bis[N-(3-chloropropyl)phenethylamine]
(5b). A mixture of 5a (141 mg, 0.22 mmol), 25% DMC-CH2Cl2
solution (0.4 ml, 0.6 mmol) and TEA (0.1 ml, 0.34 mmol) in
CH2Cl2 (5 ml) was stirred at room temperature for 24 h under N2
atmosphere. The mixture was washed with H2O, dried over
Na2SO4, and the solvent was evaporated under reduced pressure.
Purification by column chromatography on silica gel with
CHCl3:MeOH (10:1) as an eluent afforded 138 mg, 94% as a
pale yellow oil, which was used for the next reaction without
N,N'-Bis(phenethyl)-6H,12H-5,11-methanodibenzo[b,f][1,5]-
diazocine-2,8-diethanamine (3b). A mixture of 3a (200 mg,
0.37 mmol) in THF (10 ml) was stirred at room temperature
.
under N2 atmosphere. BH3 SMe3 (0.44 mL, 4.4 mmol) was
1
added and the reaction mixture then stirred for 24 h at 80 °C and
cooled to room temperature. Hydrogen chloride-MeOH (0.7M)
solution (2.2 ml) was added, and the reaction mixture was
refluxed for 1 h, and evaporated under reduced pressure. The
residue was made basic to pH 11 using an excess of 25%
NH4OH. The mixture was then extracted with CH2Cl2 (10 ml
×3). The combined organic phases were washed with brine and
dried over Na2SO4. Removal of solvent under reduced pressure
afforded a yellow oil, which was chromatographed on a silica
gel column with CHCl3:MeOH (10:1) and CHCl3:MeOH:25%
NH4OH (100:20:2) as the eluent to give a viscous oil (182 mg,
96%), which was used for the next reaction without further
further purification. H nmr (CDCl3) δ : 1.57 (4H, br s), 1.76—
2.72 (4H, br s), 2.61—2.78 (16H, m), 3.33—3.37 (4H, m), 4.10
(2H, d, J =16.6 Hz), 4.29 (2H, s), 4.65 (2H, d, J =16.6 Hz), 6.66
(2H, d, J =1.8 Hz), 6.94 (2H, dd, J =8.0, 1.8 Hz), 7.04 (2H, d, J
=8.0 Hz), 7.13—7.20 (6H, m), 7.24—7.28 (4H, m). HR-ms
(FAB) m/z: 669.3491(M+1)+, 671.3461 (M+1)+37Cl, 673.3382
(M+1)+37Cl
(Calcd for C41H51N4Cl2: 669.3490 (M+1) ,
+
2
671.3461 (M+1)+37Cl, 673.3431 (M+1)+37Cl2)
.
N,N'-(6H,12H-5,11-Methanodibenzo[b,f][1,5]diazocine-2,8-
diyldi-2,1-ethanediyl)-bis[N-(3-(4-dimethylaminopyridin-
ium)propyl)phenethylamine] dichloride (1). A mixture of 5b
(90 mg, 0.134 mmol) and 4-dimethylaminopyridine (33 mg,
0.27 mmol) in MeOH (3 ml) was stirred at 80 °C for 24 h. After
removal of the solvent under reduced pressure, the residue was
triturated with EtOAc, and the resulting hygroscopic solid was
1
purification. H-nmr (CDCl3) δ : 2.65 (4H, t, J =7.1 Hz), 2.75-
2.88 (12H, m), 4.09 (4H, d, J =16.8 Hz), 4.27 (2H, s), 4.63 (2H,
d, J =16.8 Hz), 6.69 (2H, d, J =1.7 Hz), 6.95 (2H, dd, J =8.0, 1.7