T. Schla¨ger et al. / Bioorg. Med. Chem. 16 (2008) 2992–3001
2999
J = 7.1 Hz, 2H, OCH2CH3), 6.38 (s, 1H, Ar-CH), 7.31
(t, J = 7.1 Hz, 1H, Phenyl-CH, para), 7.46–7.53 (m,
2H, Phenyl-CH, meta), 7.66–7.71 (m, 2H, Phenyl-CH,
ortho), 7.79 (s, 1H, Pyr-3-CH). C19H23N3O4 (357.5).
MS (EI): m/e (rel. int.) 357 [M+, 61], 342 [MꢀCH3,
70], 325 [MꢀHOCH3, 100], 297 [MꢀOCH3–CH3CH2,
41].
[MꢀOCH3, 17], 284 [MꢀBenzyl, 17], 91 [Benzyl, 93].
Anal. Calcd for C23H25N3O2 (375.5) C, 73.6; H, 6.71;
N, 11.2; found C, 73.6; H, 6.82; N, 11.1.
6.9. 10-Butyl-6-methoxy-1-phenyl-4,6-dihydrospiro[1H-
furo[3,4-c]pyrazole-4,40-piperidine] (3c)
To a solution of the secondary amine 3a (106.0 mg,
0.37 mmol) in THF (12 mL) butyl bromide (48.4 lL,
0.45 mmol) and K2CO3 (410.5 mg, 2.97 mmol) were
added. After heating of the mixture to reflux for 49 h
the suspension was filtered and the solvent was evapo-
rated. The residue was purified by fc (Ø = 3 cm,
EtOAc + 10% methanol). Yellow oil, yield 74 mg (59
%), Rf = 0.18 (EtOAc + 10% methanol). IR (neat): 3064
(CAHaromat.), 2927 (CAHaliphat.), 2804 (CAH), 1601,
1564, 1513 (C@C), 1086, 1062 (CAO), 754, 712 (CAH).
6.6.2. Compound 10. Colorless oil, yield 11 mg (12 %),
Rf = 0.22 (n-hexane/EtOAc, 5:5). IR (neat): 3416
(OAH), 2925 (CAHaliphat.), 1694 (C@O). 1H NMR
(DMSO-d6): d 1.18 (t, J = 7.1 Hz, 3H, CO2CH2CH3),
1.74–1.81 (m, 2H, N(CH2CH2)2), 1.99 (td, J = 13.0/4.6
Hz, 2H, N(CH2CH2)2), 3.09–3.25 (m, 2H, N(CH2CH2)2),
3.81–3.90 (m, 2H, N(CH2CH2)2), 4.05 (q, J = 7.0 Hz, 2H,
COOCH2CH3), 5.57 (s, 1H, OH), 7.42–7.56 (m, 5H, Phe-
nyl-CH), 7.84 (s, 1H, Pyr-3-CH), 10.17 (s, 1H, CHO).
C18H21N3O4 (343.4). MS (ESI): m/e (rel. int.) 343
[MH+, 100].
1H NMR (CDCl3):
d 0.94 (t, J = 7.3 Hz, 3H,
NCH2CH2CH2CH3), 1.30–1.41 (m, 2H, NCH2CH2
CH2CH3), 1.48–1.60 (m, 2H, NCH2CH2CH2CH3),
1.90–2.12 (m, 4H, N(CH2CH2)2), 2.38–2.50 (m, 2H,
NCH2CH2CH2CH3), 2.51–2.81 (m, 4H, N(CH2CH2)2),
3.46 (s, 3H, OCH3)), 6.16 (s, 1H, ArCH), 7.25 (t,
J = 7.4 Hz, 1H, Phenyl-CH, para), 7.41–7.44 (m, 2H, Phe-
nyl-CH, meta), 7.46 (s, 1H, Pyr-3-CH), 7.74 (d,
J = 7.7 Hz, 2H, Phenyl-CH, ortho). MS (EI): m/e (rel.
int.) 341 [M+, 5], 298 [M–Propyl, 100]. Anal. calcd. for
C20H27N3O2 (341.5) C, 70.4; H, 7.97; N, 12.3; found C,
70.3; H, 8.01; N, 12.2.
6.7. 6-Methoxy-1-phenyl-4,6-dihydrospiro[1H-furo[3,4-
c]pyrazole-4,40-piperidine] (3a)
The carbamate 9 (343 mg, 0.96 mmol) was dissolved in
dioxane/H2O (96 mL). Then a solution of KOH (2 M,
30 mL) was added and the mixture was refluxed for
18 h. After dilution with H2O the solution was extracted
with EtOAc (3·), the organic layer was dried (K2CO3), fil-
tered, concentrated in vacuo, and the residue was purified
by fc (Ø = 4 cm, methanol + 2% NH3concd). Pale yellow
solid, yield 210 mg (77 %), mp 81 ꢁC, Rf = 0.21 (metha-
nol + 2% NH3conc.). IR (neat): 3309 (NAH), 3063
(CAHaromat.), 2942 (CAHaliphat.), 2826 (CAH), 1599,
1563, 1512 (C@C), 1080, 1061 (CAO), 755, 710 (CAH).
7. Receptor binding studies
7.1. General information
1H NMR (DMSO-d6):
d
1.65–1.77 (m, 4H,
N(CH2CH2)2), 2.67–2.78 (m, 2H, N(CH2CH2)2), 2.87–
2.97 (m, 2H, N(CH2CH2)2), 3.34 (s, 3H, OCH3), 6.35 (s,
1H, ArCH), 7.30 (tt, J = 7.4/1.2 Hz, 1H, Phenyl-CH,
para), 7.45–7.52 (m, 2H, Phenyl-CH, meta), 7.66–7.71
(m, 2H, Phenyl-CH, ortho), 7.73 (s, 1H, Pyr-3-CH). MS
(EI): m/e (rel. int.) 285 [M+, 60], 270 [MꢀCH3, 100], 77
[Phenyl, 10]. Anal. Calcd for C16H19N3O2 (285.4) C,
67.4; H, 6.71; N, 14.7; found C, 67.1; H, 6.73; N, 14.5.
The guinea pig brains and rat livers were commercially
available (Harlan-Winkelmann, Borchen, Germany).
Homogenizer: Elvehjem Potter (B. Braun Biotech Inter-
national, Melsungen, Germany). Centrifuge: High-speed
cooling centrifuge model Sorvall RC-5C plus (Thermo
Fischer Scientific, Whatham, MA, USA). Filter: Printed
Filtermat Typ B (Perkin Elmer, Whatham, MA, USA),
presoaked in 0.5% aqueous polyethylenimine for 2 h at
rt before use. The filtration was carried out with a
MicroBeta FilterMate-96 Harvester (Perkin-Elmer,
Whatham, MA, USA). The scintillation analysis was
performed using Meltilex (Typ A) solid scintillator (Per-
kin-Elmer, Whatham, MA, USA). The solid scintillator
was melted on the filtermat at a temperature of 95 ꢁC for
5 min. After solidification of the scintillator at rt, the
scintillation was measured using a MicroBeta Trilux
scintillation analyzer (Perkin-Elmer, Whatham, MA,
USA). The overall counting efficiency was 20%, the
counting time 5 min. All experiments were carried out
in duplicate using standard 96-well multiplates (Diago-
6.8. 10-Benzyl-6-methoxy-1-phenyl-4,6-dihydrospiro[1H-
furo[3,4-c]pyrazole-4,40-piperidine] (3b)
To a solution of the secondary amine 3a (60.0 mg,
0.21 mmol) in THF (7 mL) benzyl bromide (29.9 lL,
0.25 mmol) and K2CO3 (232.2 mg, 1.68 mmol) were
added. After heating of the mixture to reflux for 24 h
the suspension was filtered and concentrated in vacuo.
The residue was purified by fc (Ø = 3 cm, n-hexane/
EtOAc, 5:5). Pale yellow resin, yield 56 mg (71 %),
Rf = 0.20 (n-hexane/EtOAc, 5/5). IR (neat): 3061
(CAHaromat.), 2923 (CAHaliphat.), 2804 (CAH), 1600,
1565, 1512 (C@C), 1080, 1061 (CAO), 755, 738
(CAH). 1H NMR (CDCl3): d 1.86–2.12 (m, 4H,
N(CH2CH2)2), 2.49–2.80 (m, 4H, N(CH2CH2)2), 3.46
(s, 3H, OCH3), 3.62 (s, 2H, NCH2Ph), 6.16 (s, 1H,
ArCH), 7.24–7.46 (m, 9H, aromat. CH), 7.73 (dd,
J = 8.4/0.8 Hz, 2H, Phenyl-CH, ortho). MS (EI): m/e
(rel. int.) 375 [M+, 60], 360 [MꢀCH3, 100], 344
nal, Munster, Germany). The IC values were deter-
50
¨
mined in competition experiments with at least six
concentrations of the test compounds and were calcu-
lated with the program GraphPad Prismꢂ 3.0 (Graph-
Pad Software) by non-linear regression analysis. The
Ki values were calculated according to Cheng and Prus-
off.28 The Ki values are given as mean values SEM
from three independent assays.