48
P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
8.03 (dd, 1H, J ¼ 8.7 Hz, J ¼ 1.8 Hz), 8.17e8.21 (t, 1H, J ¼ 5.9 Hz), 8.50
(br s,1H), 8.65e8.68 (d,1H, J ¼ 8.0 Hz), 9.12e9.15 (dd,1H, J ¼ 4.5 Hz,
J ¼ 1.5 Hz). C22H24Cl2N4O2S$HCl, Mp ¼ 211.1e212.9 ꢁC. Anal. Calc for
C22H24Cl2N4O2S HCl (515.88): C, 51.22; H, 4.88; N, 10.86; found C,
51.36; H, 4.89; N, 10.83.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
[M þ H]þ ¼ 493.2. 1H NMR (DMSO)
d (ppm) 1.29 (br s, 4H), 2.06e
2.10 (m, 2H), 2.33 (br s, 4H), 2.82e2.88 (m, 6H), 7.09e7.13 (m,
1H), 7.21e7.33 (m, 3H), 7.69e7.73 (m, 2H), 8.27e8.32 (m, 2H), 8.53e
8.56 (dd, 1H, J ¼ 8.5 Hz, J ¼ 1.8 Hz), 9.06e9.08 (m, 1H).
C23H26Cl2N4O2S$HCl, Mp
C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,
¼
131.1e131.9 ꢁC. Anal. Calc for
4.3.4. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
3-sulfonamide (34)
52.29; H, 5.16; N, 10.53.
Yellow solid, 344 mg (70% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.38.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
4.3.9. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)
isoquinoline-3-sulfonamide (39)
White solid, 334 mg (68% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.33.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
[M þ H]þ ¼ 493.1. 1H NMR (DMSO)
d
(ppm) 1.35e1.37 (m, 4H), 2.17e
2.25 (m, 2H), 2.34 (br s, 4H), 2.84e2.87 (m, 6H), 7.04e7.09 (m, 1H),
7.26e7.32 (m, 2H), 7.72e7.77 (m, 1H), 7.90e7.95 (m, 2H), 8.11e8.13
(m, 1H), 8.22e8.25 (m, 1H), 8.86 (s, 1H), 9.17 (s, 1H).
[M þ H]þ ¼ 492.7. 1H NMR (CDCl3)
d (ppm) 1.58e1.60 (m, 4H),
C23H26Cl2N4O2S$HCl, Mp
C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,
¼
148.0e149.7 ꢁC. Anal. Calc for
2.42e2.44 (m, 2H), 2.72 (br s, 4H), 3.03 (br s, 2H), 3.18e3.21 (m, 4H),
6.99e7.04 (m, 1H), 7.08e7.16 (m, 2H), 7.74e7.85 (m, 2H), 7.94e7.97
(d, 1H, J ¼ 8.0 Hz), 8.04e8.07 (dd, 1H, J ¼ 7.82 Hz, J ¼ 0.8 Hz), 8.43 (s,
1H), 8.65 (br s, 1H), 9.26 (s, 1H). C23H26Cl2N4O2S$HCl, Mp ¼ 185.3e
186.7 ꢁC. Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H,
5.14; N, 10.57; found C, 52.03; H, 5.12; N, 10.59.
52.23; H, 5.15; N, 10.53.
4.3.5. 6-Chloro-N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)
quinoline-4-sulfonamide (35)
Yellow solid, 305 mg (58% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.54.
C23H25Cl3N4O2S, MW 527.89, monoisotopic mass 526.08,
4.3.10. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)
isoquinoline-4-sulfonamide (40)
Yellow oil, 344 mg (70% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.28.
C23H26Cl2N4O2S, MW 493.45, Monoisotopic Mass 492.12,
[M þ H]þ ¼ 527.2. 1H NMR (DMSO)
d
(ppm) 1.43e1.50 (m, 2H), 1.72
(m, 2H), 2.84e2.90 (m, 2H), 3.03e3.24 (m, 6H), 3.35e3.50 (dd, 4H,
J ¼ 31.0 Hz, J ¼ 12.0 Hz), 7.16e7.19 (m, 1H), 7.33e7.35 (m, 2H), 7.93e
7.97 (dd, 1H, J ¼ 9.0 Hz, J ¼ 2.3 Hz), 8.13e8.16 (d, 1H, J ¼ 9.23 Hz),
8.18e8.20 (m, 1H), 8.41e8.42 (d, 1H, J ¼ 2.3 Hz), 8.88e8.89 (d, 1H,
J ¼ 2.0 Hz), 9.21e9.22 (d, 1H, J ¼ 2.3 Hz). C23H25Cl3N4O2S$HCl,
Mp ¼ 191.2e192.7 ꢁC. Anal. Calc for C23H25Cl3N4O2S$HCl (564.35):
C, 48.95; H, 4.64; N, 9.93; found C, 48.81; H, 4.62; N, 9.95.
[M þ H]þ ¼ 493.0. 1H NMR (CDCl3)
d (ppm) 1.61 (br s, 4H), 2.42e
2.46 (t, 2H, J ¼ 5.1 Hz), 2.72 (br s, 4H), 3.03e3.06 (m, 2H), 3.19e
3.22 (t, 4H, J ¼ 4.5 Hz), 7.02e7.05 (q, 1H, J ¼ 3.2 Hz), 7.15e7.19 (m,
2H), 7.70e7.77 (m, 1H), 7.80e7.86 (m, 2H), 8.08e8.10 (d, 1H,
J ¼ 7.7 Hz), 8.70e8.73 (d, 1H, J ¼ 9.2 Hz), 9.11 (s, 1H), 9.39 (s, 1H).
C23H26Cl2N4O2S$HCl, Mp
¼
145.1e147.4 ꢁC. Anal. Calc for
4.3.6. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
6-sulfonamide (36)
C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,
52.16; H, 5.13; N, 10.53.
Yellow solid, 305 mg (62% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.20.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
4.4. In vitro pharmacology
[M þ H]þ ¼ 493.2. 1H NMR (DMSO)
d
(ppm) 1.38e1.46 (m, 2H),
4.4.1. Cell culture
1.64e1.75 (br s, 2H), 2.79e2.84 (q, 2H, J ¼ 6.4 Hz), 3.04e3.16 (m,
6H), 3.37e3.49 (m, 4H), 7.17e7.20 (m, 1H), 7.33e7.36 (m, 2H), 7.81e
7.86 (m, 1H), 8.02e8.08 (m, 1H), 8.16e8.20 (dd, 1H, J ¼ 8.9 Hz,
J ¼ 2.1 Hz), 8.32e8.38 (d, 1H, J ¼ 9.0 Hz), 8.63e8.64 (d, 1H,
J ¼ 1.8 Hz), 8.82e8.88 (d, 1H, J ¼ 7.7 Hz), 9.15e9.18 (dd, 1H,
J ¼ 4.6 Hz, J ¼ 1.5 Hz). C23H26Cl2N4O2S$HCl, Mp ¼ 147.1e148.8 ꢁC.
Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N,
10.57; found C, 52.31; H, 5.14; N, 10.60.
All receptor cDNAs were obtained from the Missouri S&T cDNA
Resource Center (www.cdna.org). Human serotonin 5-HT2AR cDNA
was cloned into the pcDNA5/FRT/TO vector (Invitrogen) and
transfected into the HEK293 cells. Cell line with tetracycline-
dependent expression of 5-HT2AR was developed using the FLP-
IN T-REx system according to manufacturers protocols (Invi-
trogen). HEK293 cells with stable expression of human serotonin 5-
HT1AR, 5-HT6, 5-HT7bR or dopamine D2LR were obtained with the
use of Lipofectamine 2000 (Invitrogen). Cells were maintained at
37 ꢁC in a humidified atmosphere with 5% CO2 and were grown in
Dulbecco’s Modified Eagle Medium containing 10% dialyzed fetal
4.3.7. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
7-sulfonamide (37)
Yellow solid, 319 mg (65% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.27.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
bovine serum under selective conditions (75
m
g/ml hygromycin and
10 g/ml blasticidin for the 5-HT2AR cell line and 500
m
mg/ml G418
sulfate for the other cells). 48 h prior to cell culture harvest the
expression of the 5-HT2A receptor was induced by addition of the
2 mg/ml tetracycline. For membranes preparations, cells were
[M þ H]þ ¼ 493.0. 1H NMR (CDCl3)
d (ppm) 1.62 (br s, 4H), 2.42 (br s,
2H), 2.69 (br s, 4H), 3.06 (br s, 2H), 3.16e3.19 (t, 4H, J ¼ 4.6 Hz),
7.03e7.06 (m, 1H), 7.16e7.18 (m, 2H), 7.51e7.56 (q, 1H, J ¼ 4.1 Hz),
7.95 (m, 3H), 8.20e8.23 (m, 1H), 8.63 (s, 1H), 9.02e9.04 (dd, 1H,
J ¼ 4.4 Hz J ¼ 1.8 Hz). C23H26Cl2N4O2S$HCl, Mp ¼ 142.8e144.1 ꢁC.
Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N,
10.57; found C, 51.94; H, 5.13; N, 10.60.
subcultured into 150 cm2 cell culture flasks, grown to 90% conflu-
ence, washed twice with prewarmed to 37 ꢁC phosphate buffered
saline (PBS) and were pelleted by centrifugation (200 g) in PBS
containing 0.1 mM EDTA and 1 mM dithiothreitol, and stored
at ꢂ80 ꢁC.
4.3.8. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
8-sulfonamide (38)
White solid, 255 mg (52% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5);tR ¼ 1.37.
4.4.2. 5-HT1A/5-HT2A/5-HT6/5-HT7/D2L radioligand binding assays
Membrane preparation and general assay procedures for cloned
receptors were adjusted to 96-microwell format based on protocols
was described by us previously [4,23,24].