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of the crude. The desired products were purified by FC, and enan-
tiomeric excess was determined by chiral HPLC.
141.2, 139.6, 128.9, 127.6, 127.4, 127.3, 127.2, 41.5, 36.9, 26.1, 24.6,
21.8 ppm; IR (neat): MS (ESI): m/z 308.22 [M+H]+.
(+)-(S)-Ethyl 3-(biphenyl-4-yl)butanoate [(+)-(S)-4]: Purification by
FC (n-hexane/EtOAc 97:3) gave the desired product as a yellow oil
(739 mg, 95%): 87% ee determined by analytical chiral HPLC
[Daicel Chiralcel OJ-H (1=0.46 cm, l=15 cm, 5 mm), n-heptane/ 2-
(+)-(S)-1-(3-(Biphenyl-4-yl)butyl)piperidine [(+)-(S)-RC-33]: A so-
lution of (+)-(S)-7 (61 mg, 0.2 mmol) in anhydrous THF (5 mL) was
added dropwise to
a stirred suspension of LiAlH4 (23 mg,
0.6 mmol) in anhydrous THF (6 mL). The reaction mixture was
stirred at RT for 3 h, cooled to 08C, slowly quenched with H2O,
stirred for 15 min at RT and finally filtered on a pad of Celite (wash-
ing with EtOAc). The filtrate was evaporated, the residue dissolved
in EtOAc (30 mL) and extracted with H2O (3ꢃ20 mL). The organic
phase was dried over anhydrous Na2SO4 and evaporated, yielding
the desired compound as a yellow oil (53 mg, 90%): 87% ee deter-
mined by analytical chiral HPLC [Daicel Chiralcel OJ-H (1=
0.46 cm, l=15 cm, 5 mm), CH3OH/Et3N 100:0.1, flow rate=
propanol 90:10, flow rate=0.8 mLminꢁ1
, l=250 nm]: tRmajor =
8.69 min,
t
Rminor =11.24 min; [a]D20 = +29.5 (c=0.5 in CHCl3);
3
1H NMR (400 MHz, CDCl3): d=7.58 (m, 2H), 7.54 (d, J(H,H)=8.3 Hz,
2H), 7.43 (t, 3J(H,H)=7.6 Hz, 2H), 7.34 (m, 1H), 7.31 (d, 3J(H,H)=
8.3 Hz, 2H), 4.10 (q, J(H,H)=7.1 Hz, 2H), 3.34 (m, 1H), 2.66 (dd, AB
3
system, 2J(H,H)=15 Hz, 3J(H,H)=7.1 Hz, 1H), 2.58 (dd, AB system,
2J(H,H)=15 Hz, 3J(H,H)=8.1 Hz, 1H), 1.35 (d, 3J(H,H)=7.0 Hz, 3H),
1.20 ppm (t, 3J(H,H)=7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=
172.6, 145.1, 141.2, 139.5, 128.9, 127.4, 127.3, 127.2, 60.5, 43.2, 36.4,
22.0, 14.4 ppm; MS (ESI): m/z: 269.12 [M+H]+.
0.5 mLminꢁ1
,
l=250 nm]:
t
Rmajor =9.91 min,
tRminor =11.44 min;
[a]D20 = +19.2 (c=1.0 in CH3OH); MS (ESI): m/z 294.25 [M+H]+.
Spectroscopic properties comply with those reported above for
(+)-(S)-RC-33 obtained by semi-preparative HPLC.
(+)-(S)-Ethyl 3-phenylbutanoate [(+)-(S)-5]: Purification by FC (n-
hexane/EtOAc 98:2) gave the desired product as colorless oil
(546 mg, 98%): 87% ee determined by analytical chiral HPLC
[Daicel Chiralcel OJ-H (1=0.46 cm, l=15 cm, 5 mm), n-heptane/ 2-
Circular dichroism: Solutions of (+)-4 (c=6.25ꢃ10ꢁ5 m in n-
hexane, optical pathway 1 cm) and of (+)-(S)-5 (c=2.5ꢃ10ꢁ5 m in
n-hexane, optical pathway 1 cm) were analyzed in a nitrogen at-
mosphere. CD spectra were scanned at 10 nmminꢁ1 with a spectral
band width of 2 nm and data resolution of 0.2 nm. Experimental
data are reported in the Supporting Information.
propanol 90:10, flow rate=0.8 mLminꢁ1
, l=250 nm]: tRminor =
5.36 min, tRmajor =5.91 min; [a]D20 = +18.3 (c=0.5 in CHCl3). Spec-
troscopic properties comply with those reported earlier.[10]
(+)-(S)-3-(Biphenyl-4-yl)butanoic acid [(+)-(S)-6]: To a solution of
(+)-(S)-4 (456 mg, 1.7 mmol) in EtOH (23 mL), 2.0m NaOH (23 mL)
was added. The mixture was stirred at RT for 2 h, concentrated
under vacuum, adjusted to pH 2 with HCl (1.0m) and extracted
with CH2Cl2 (3ꢃ40 mL). The combined organic phases were dried
over anhydrous Na2SO4 and evaporated to dryness, yielding the de-
sired product as a white solid (401 mg, 98%): 87% ee determined
by analytical chiral HPLC [Daicel Chiralpak IC (1=0.46 cm, l=
15 cm, 5 mm), n-heptane/2-propanol/TFA 96:4:0.1, flow rate=
0.8 mLminꢁ1, l=250 nm]: tRmajor =8.31 min, tRminor =9.85 min; mp:
110–1128C; [a]D20 = +33.6 (c=1.0 in CH3OH); 1H NMR (400 MHz,
[D6] acetone): d=10.6 (brs, 1H), 7.64 (m, 2H), 7.59 (d, 3J(H,H)=
8.3 Hz, 2H), 7.44 (t, 3J(H,H)=7.6 Hz, 2H), 7.39 (d, 3J(H,H)=8.2 Hz,
2H), 7.34 (m, 1H), 3.29 (m, 1H), 2.70–2.58 (m, 2H), 1.33 ppm (d,
3J(H,H)=7.0 Hz, 3H); 13C NMR (100 MHz, [D6] acetone): d=173.4,
146.3, 141.7, 139.8, 129.6, 128.2, 128.0, 127.7, 127.6, 42.6, 36.8,
22.4 ppm; MS (ESI): m/z 239.11 [MꢁH]ꢁ.
Biological investigations
In vitro binding assays
Materials: Guinea pig brains, rat liver, and rat brains for the s1, s2,
m-, k-, and d-opioid receptor binding assays were commercially
available (Harlan–Winkelmann, Borchen, Germany). Pig brains for
binding assays to the PCP binding site of the NMDA receptor were
a kind donation from a local slaughterhouse (Coesfeld, Germany).
Homogenizer: Elvehjem Potter (B. Braun Biotech International, Mel-
sungen, Germany) and Soniprep 150, MSE, London, UK). Centrifug-
es: Cooling centrifuge model Rotina 35R (Hettich, Tuttlingen, Ger-
many) and High-speed cooling centrifuge model Sorvall RC-5C plus
(Thermo Fisher Scientific, Langenselbold, Germany). Multiplates:
standard 96-well multiplates (Diagonal, Muenster, Germany).
Shaker: self-made device with adjustable temperature and tum-
bling speed (scientific workshop of the institute). Vortexer: Vortex
Genie 2 (Thermo Fisher Scientific, Langenselbold, Germany). Har-
vester: MicroBeta FilterMate-96 Harvester. Filter: Printed Filtermat
Type A and B. Scintillator: Meltilex (Type A or B) solid-state scintilla-
tor. Scintillation analyzer: MicroBeta Trilux (all PerkinElmer LAS,
Rodgau-Jꢀgesheim, Germany). Chemicals and reagents were pur-
chased from various commercial sources and were of analytical
grade.
(+)-(S)-3-(Biphenyl-4-yl)-1-(piperidin-1-yl)butan-1-one [(+)-(S)-7]:
In a microwave vial, TBTU (321 mg, 1 mmol) was added to a solu-
tion of (+)-(S)-6 (120 mg, 0.5 mmol) in THF (16 mL). The mixture
was stirred at RT for 5 min, and then a solution of N,N-diisopropyl-
ethylamine (DIPEA, 0.2 mL, 1 mmol) and piperidine (0.1 mL,
1 mmol) in THF (2 mL) was added. The reaction mixture was irradi-
ated by microwave at 25 W, 808C for 20 min, and then the solvent
was evaporated under vacuum. The residue was then dissolved
with CH2Cl2 (30 mL), and the organic phase was washed with 0.5m
HCl (3ꢃ15 mL), dried over anhydrous Na2SO4, and evaporated,
yielding the desired product as a yellow oil (147 mg, 96%): 87% ee
determined by analytical chiral HPLC (Daicel Chiralcel OJ-H (1=
0.46 cm, l=15 cm, 5 mm), n-heptane/2-propanol 90:10, flow rate=
Preparation of membrane homogenates from guinea pig brain: Five
guinea pig brains were homogenized with the potter (500–
800 rpm, 10 up-and-down strokes) in six volumes of cold 0.32m su-
crose. The suspension was centrifuged at 1200 g for 10 min at 48C.
The supernatant was separated and centrifuged at 23500 g for
20 min at 48C. The pellet was resuspended in 5–6 volumes of
buffer (50 mm Tris, pH 7.4) and centrifuged again at 23500 g
(20 min, 48C). This procedure was repeated twice. The final pellet
was resuspended in 5–6 volumes of buffer and frozen (ꢁ808C) in
0.8 mLminꢁ1
[a]D20 = +11.6 (c=0.5 in CH3OH); 1H NMR (400 MHz, CDCl3): d=
, l=250 nm): tRmajor =10.09 min, tRminor =12.03 min;
3
3
7.57 (m, 2H), 7.53 (d, J(H,H)=8.2 Hz, 2H), 7.43 (t, J(H,H)=7.6 Hz,
2H), 7.35–7.31 (m, 3H), 3.59–3.29 (m, 5H), 2.67 (dd, AB system,
2J(H,H)=14.7 Hz, 3J(H,H)=6.5 Hz, 1H), 2.56 (dd, AB system,
3
2J(H,H)=14.7 Hz, J(H,H)=7.9 Hz, 1H), 1.59–1.47 (m, 6H), 1.38 ppm
1.5 mL portions containing ~1.5 (mg protein)mLꢁ1
.
3
(d, J(H,H)=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=170.8, 145.5,
Preparation of membrane homogenates from rat liver: Two rat livers
(Sprague–Dawley rats) were cut into small pieces and homogen-
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 1514 – 1527 1522