CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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mixture was hydrogenated in a high pressure microwave reactor [11] at 2708 and 60 bar H2 pressure for
4.5 h (heating rate, 10 min 258 to 2708). After cooling to 258, the catalyst was filtered off, and the solvent
was evaporated. The residue was treated with 150 ml of 2.0m NaOH, extracted with CH2Cl2 (3ꢁ120 ml),
and dried (MgSO4). The solvent was evaporated, and the residue was purified by CC (SiO2; (Rf (CHCl3/
MeOH/25% aq. NH3 100 :10 :1) 0.10) to give 10 (1.05 g, 52%). Colorless foam.. [a]D23 ¼ ꢂ23.08 (c¼2.0,
1
CHCl3) ([17]: [a]2D3 ¼ ꢂ24.5, CHCl3). H-NMR (CDCl3, 400 MHz): 8.11 (d, J¼7.9, HꢂC(4)); 7.25–7.15
(m, HꢂC(1), HꢂC(3)); 7.10 (t, J¼7.4, HꢂC(2)); 4.06 (d, J¼5.5, HꢂC(8)); 3.68 (t, J¼6.3, C(23)); 3.09 (td,
J¼11.9, 7.2, HaꢂC(11)); 2.99 (dd, J¼11.9, 6.9, HbꢂC(11)); 2.92 (dd, J¼10.7, 3.6, HaꢂC(20)); 2.88 (m,
HꢂC(16)); 2.49 (m, C(18)); 2.27–2.43 (m, C(12), HbꢂC(21)); 2.07–2.18 (m, HaꢂC(17), HꢂC(13)); 1.95
(m, HꢂC(21)); 1.65–1.80 (m, HꢂC(14), CH2(15), HbꢂC(17)); 1.53 (m, HaꢂC(22)); 1.43 (m, HbꢂC(22)).
13C-NMR (CDCl3, 100 MHz): 169.2 (C¼O); 141.1 (C(5)); 138.8 (C(6)); 127.9 (C(3)); 125.1 (C(2)); 122.0
(C(1)); 117.7 (C(4)); 67.5 (C(8)); 67.0 (C(16)); 60.2 (C(23)); 55.3 (C(22)); 52.8 (C(7)); 50.4 (C(11)); 37.8
(C(12)); 37.7 (C(21)); 34.7 (C(22)); 32.1 (C(14)); 29.7 (C(18)); 28.7 (C(17)); 26.8 (C(13)); 26.6 (C(15)).
21,22-Dihydroisostrychnine (¼(3aR,11bS,12S)-14-(2-Hydroxyethyl)-2,3,10,12,13,13a-hexahydro-
9H,11bH-1,12-ethanopyrido[1,2,3-lm]pyrrolo[2,3-d]carbazol-9-one; 9). Compound 9 (100 mg, 5%) was
obtained as a minor fraction during the purification of 10 by CC (SiO2; Rf (CHCl3/MeOH/25% aq. NH3
100 :10 :1) 0.15). White solid. M.p. 243–2458 ([17]: 244–2468). 1H-NMR (CDCl3, 400 MHz): 8.10 (d, J¼
7.7, HꢂC(4)); 7.24–7.15 (m, HꢂC(1), HꢂC(3)); 7.07 (td, J¼7.5, 1.0, HꢂC(2)); 5.75 (m, HꢂC(12)); 4.25 (t,
J¼2.9, HꢂC(8)); 3.71 (td, J¼6.4, 2.0, C(23)); 3.39 (t, J¼3.0, HꢂC(16)); 3.24–2.90 (m, HaꢂC(11),
HaꢂC(18), HbꢂC(21), HaꢂC(20), HbꢂC(11)); 2.53 (m, HꢂC(14)); 2.45 (m, HaꢂC(7)); 2.30 (ddd, J¼13.9,
9.0, 1.5, HbꢂC(17)); 2.14–2.03 (m, HaꢂC(15), HꢂC(21)); 1.79 (t, J¼12.1, HbꢂC(20)); 1.59–1.48 (m,
HaꢂC(22), HbꢂC(15)); 1.35 (dddd, J¼13.5, 6.7, 6.7, 6.7, HbꢂC(22). 13C-NMR (CDCl3, 100 MHz): 168.7
(C¼O); 141.2 (C(5)); 141.1 (C(13)); 136.3 (C(6)); 128.2 (C(3)); 124.4 (C(2)); 122.4 (C(12)); 122.3 (C(1));
114.4 (C(4)); 70.8 (C(8)); 65.4 (C(16)); 60.0 (C(23)); 54.6 (C(11)); 53.7 (C(20)); 51.7 (C(7)); 48.7
(C(17)); 37.3 (C(18)); 36.7 (C(14)); 36.1 (C(21)); 35.4 (C(22)); 27.4 (C(15)). EI-MS: 337.1 (23), 336.1 (78,
Mþ ), 316.1 (30), 305.1 (33), 291.1 (42), 220.1 (45), 167.1 (49), 149.0 (69).
23-Deoxy-12,13,21,22-tetrahydroisostrychnine (¼(3aR,11aR,11bS,12S)-14-Ethyl-2,3,10,11,11a,
12,13,13a-octahydro-9H,11bH-1,12-ethanopyrido[1,2,3-lm]pyrrolo[2,3-d]carbazol-9-one; 11). Com-
pound 11 (75 mg, 4%) was obtained as a minor fraction during the purification of 10 by CC (SiO2; Rf
(CHCl3/MeOH/25% aq. NH3 100 :10 :1) 0.49). White foam. M.p. 125–1308 ([18]: 174–1768). 1H-NMR
(CDCl3, 400 MHz): 8.10 (d, J¼7.8, HꢂC(4)); 7.23–7.15 (m, HꢂC(1), HꢂC(3)); 7.09 (td, J¼7.4, 1.0,
HꢂC(2)); 4.04 (d, J¼5.5, HꢂC(8)); 3.09 (m, HaꢂC(11)); 2.99 (m, HbꢂC(11)); 2.92–2.86 (m, HaꢂC(20),
HꢂC(16)); 2.53–2.47 (m, CH2(18)); 2.40–2.26 (m, CH2(12), HbꢂC(20)); 2.16–2.06 (m, HaꢂC(17),
HꢂC(14)); 1.75–1.61 (m, CH2(15), HꢂC(13), HbꢂC(17), HꢂC(21)); 1.31–1.17 (m, CH2(22)); 0.93 (t, J¼
7.4, Me(23)). 13C-NMR (CDCl3, 100 MHz): 168.2 (C¼O); 140.2 (C(5)); 138.0 (C(6)); 126.8 (C(23));
124.0 (C(2)); 121.0 (C(1)); 116.7 (C(4)); 66.6 (C(8)); 66.2 (C(16)); 54.3 (C(11)); 51.9 (C(7)); 49.3
(C(20)); 41.6 (C(13)); 36.8 (C(12)); 30.2 (C(21)); 28.7 (C(18)); 27.6 (C(15)); 22.7 (C(17)); 22.5 (C(14));
23.3 (C(22)); 10.8 (Me(23)). EI-MS: 323.1 (10), 322.1 (44, Mþ ), 316.1 (30), 167.0 (36), 149.0 (100), 124.1
(65).
FLIPRꢄ Membrane Potential Blue Assay. The functional characterization of the strychnine analogs
were performed by the FLIPRꢄ membrane potential blue assay (Molecular Devices) [3]. TsA-201 Cells
transiently transfected with cDNAs encoding for a1 or a1b GlyRs were split into poly-d-lysine-coated
black 96-wells plates with clear bottom (BD Biosciences, Bedford, MA). Later (16–24 h), the medium
was aspirated, and the cells were washed with 100 ml of Krebs buffer (140 mm NaCl/4.7 mm KCl/2.5 mm
CaCl2/1.2 mm MgCl2/11 mm HEPES/10 mm d-glucose; pH 7.4). Krebs buffer (50 ml) was added to the
wells (in the antagonist experiments, various concentrations of the antagonist were dissolved in the
buffer), and then an additional 50 ml of Krebs buffer supplemented with the FMP assay dye (1 mg/ml)
was added to each well. Then, the plate was incubated at 378 in a humidified 5% CO2 incubator for 30 min
and assayed in a NOVOstarꢄ plate reader (BMG Labtechnologies, DE-Offenburg), measuring emission
(in fluorescence units (FU)) at 560 nm caused by excitation at 530 nm before and up to 1 min after
addition of 33 ml of agonist soln. The experiments were performed in duplicate at least three times for
each compound at each receptor using an EC50 concentration of glycine as agonist.