Journal of Medicinal Chemistry
Article
4H), 1.50−1.46 (m, 4H), 1.44−1.28 (m, 20H), 0.89 (t, J = 6.4 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ 159.9, 144.0, 126.0, 39.8, 34.6,
32.0, 29.5, 29.4, 27.7, 22.8, 14.2 ppm. HPLC purity: 100% (tR 10.21
min). ESI-MS: 346.40 [M].
2,6-Di(4-(4-methylphenyl)phenylethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (43). Prepared from S34c and purified by
FCC (dichloromethane:methanol 30:1 to 5:1) to give the desired
product. Yield (27 mg) 48%. 1H NMR (400 MHz, CDCl3): δ 8.43 (t, J
= 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.4 Hz, 4H), 7.67
(d, J = 8.4 Hz, 4H), 7.52 (d, J = 8.0 Hz, 4H), 7.29−7.26 (m, 4H), 4.68
(s, 3H), 2.41 (s, 6H). 13C NMR (101 MHz, CDCl3/MeOD): δ 144.9,
143.9, 139.4, 138.8, 136.3, 133.4, 133.3, 130.6, 129.8, 127.4, 127.0,
117.0, 109.1, 80.9, 77.3, 21.1 ppm. HPLC purity: 99% (tR 10.28 min).
ESI-MS: 474.40 [M].
2,6-Di([4-(4-methoxyphenyl)phenyl]ethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (44). Prepared from S34d and purified by
FCC (dichloromethane:methanol 30:1 to 10:1) to give the desired
product. Yield (42 mg) 58%. 1H NMR (400 MHz, CDCl3): δ 8.42 (t, J
= 8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.4 Hz, 4H), 7.65
(d, J = 8.4 Hz, 4H), 7.57 (d, J = 8.8 Hz, 4H), 7.00 (d, J = 8.8 Hz, 4H),
4.67 (s, 3H), 3.86 (s, 6H). 13C NMR (101 MHz, CDCl3/MeOD): δ
160.1, 144.5, 143.7, 139.3, 133.3, 131.6, 130.4, 128.3, 127.0, 116.5,
114.5, 109.3, 80.7, 55.4, 44.9 ppm. HPLC purity: 100% (tR 9.78 min).
ESI-MS: 506.33 [M].
2,6-Di([4-(4-hydroxyphenyl)phenyl]ethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (45). 44 (27 mg, 0.04 mmol) was
dissolved in 2 mL of CH2Cl2 and cooled to −78 °C under a nitrogen
atmosphere. An 1 M solution of BBr3 in CH2Cl2 (0.4 mL) was added
and the reaction mixture was allowed to warm up to room temperature
and stirring was continued for 2 h at room temperature, after which as
shown by TLC (dichloromethane:methanol 30:1) full conversion was
reached. The reaction mixture was recooled to −78 °C and quenched
with H2O. After an hour, the precipitate was filtered off and washed
with H2O and CH2Cl2, respectively, and dried in vacuo. Yield (12 mg)
48%. 1H NMR (400 MHz, DMSO): δ 9.81 (s, 2H), 8.56 (t, J = 8.4 Hz,
1H), 8.36 (d, J = 7.6 Hz, 2H), 7.89 (d, J = 8.0 Hz, 4H), 7.83 (d, J = 8.4
Hz, 4H), 7.64 (d, J = 8.4 Hz, 4H), 6.90 (d, J = 8.8 Hz, 4H), 4.58 (s,
3H). 13C NMR (151 MHz, DMSO): δ 158.2, 143.6, 143.2, 133.3,
130.6, 129.1, 128.2, 126.3, 116.4, 115.9, 106.0, 81.7, 45.2 ppm. HPLC
purity 99% (tR 8.38 min). ESI-MS: 478.33 [M].
2,6-Di([4-(4-chlorophenyl)phenyl]ethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (46). Prepared from S34e and purified by
FCC (dichloromethane:methanol 30:1 to 10:1) to give the desired
product. Yield (42 mg) 58%. 1H NMR (400 MHz, CDCl3): δ 8.43 (t, J
= 8.0 Hz, 1H), 8.08 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 4H), 7.67
(d, J = 8.4 Hz, 4H), 7.56 (d, J = 8.4 Hz, 4H), 7.46 (d, J = 8.4 Hz, 4H),
4.74 (s, 3H). 13C NMR (101 MHz, CDCl3/MeOD): δ 143.8, 143.6,
139.3, 137.7, 134.8, 133.4, 130.6, 129.2, 128.4, 127.4, 117.6, 108.6,
80.9, 45.0 ppm. HPLC purity: 99% (tR 10.39 min). ESI-MS: 514.27
[M].
2,6-Didecyl-1-methylpyridinium Trifluoromethanesulfonate (28).
Prepared from S24d and purified by FCC (dichloromethane:methanol
1
60:1 to 10:1) to give the desired product. Yield (166 mg) 74%. H
NMR (400 MHz, CDCl3): δ 8.18 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0
Hz, 2H), 4.22 (s, 3H), 3.13 (t, J = 8.0 Hz, 4H), 1.82−1.74 (m, 4H),
1.50−1.44 (m, 4H), 1.39−1.28 (m, 24H), 0.89 (t, J = 6.8 Hz, 6H). 13C
NMR (101 MHz, CDCl3): δ 159.7, 144.0, 125.9, 39.6, 34.4, 31.9, 29.5,
29.4, 29.3, 29.2, 27.6, 22.7, 14.1 ppm. HPLC purity: 100% (tR 10.77
min). ESI-MS: 374.40 [M].
2,6-Didodecyl-1-methylpyridinium Trifluoromethanesulfonate
(29). Prepared from S24e and purified by FCC (dichloromethane:-
methanol 30:1 to 20:1) to give the desired product. Yield (536 mg)
87%. 1H NMR (400 MHz, CDCl3): δ 8.19 (t, J = 8.0 Hz, 1H), 7.63 (d,
J = 7.6 Hz, 2H), 4.21 (s, 3H), 3.12 (t, J = 8.0 Hz, 4H), 1.81−1.73 (m,
4H), 1.51−1.44 (m, 4H), 1.38−1.27 (m, 32H), 0.88 (t, J = 6.8 Hz,
6H). 13C NMR (101 MHz, CDCl3): δ 159.8, 144.1, 126.0, 39.7, 34.6,
32.0, 29.8, 29.7, 29.7, 29.6, 29.5, 29.4, 29.3, 27.7, 22.8, 14.2 ppm.
HPLC purity: 100% (tR 11.84 min). ESI-MS: 430.53 [M].
2,6-Di(dodec-1-ynyl)-1-methylpyridinium Trifluoromethanesulfo-
nate (30). Prepared from S23e and purified by FCC (dichlorome-
thane:methanol 40:1 to 20:1) to give the desired product. Yield (675
1
mg) 94%. H NMR (400 MHz, CDCl3): δ 8.38 (t, J = 8.0 Hz, 1H),
7.90 (d, J = 8.0 Hz, 2H), 4.49 (s, 3H), 2.65 (t, J = 7.2 Hz, 4H), 1.74−
1.65 (m, 4H), 1.48−1.41 (m, 4H), 1.30−1.27 (m, 24H), 0.88 (t, J =
6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 144.3, 139.5, 130.7,
112.8, 72.9, 45.1, 32.0, 29.7, 29.5, 29.4, 29.2, 29.1, 27.6, 22.9, 20.3, 14.2
ppm. HPLC purity: 100% (tR 11.57 min). ESI-MS: 422.40 [M].
2,6-Di([4-(2-methylphenyl)phenyl]ethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (39). Prepared from S34a and purified by
FCC (dichloromethane:methanol 30:1 to 20:1) and preparative
HPLC to give the desired product. Yield (1 mg) 96%. 1H NMR
(400 MHz, CDCl3): δ 8.47 (t, J = 8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz,
2H), 7.77 (d, J = 8.0 Hz, 4H), 7.46 (d, J = 8.0 Hz, 4H), 7.31−7.09 (m,
8H), 4.74 (s, 3H), 2.30 (s, 6H). HPLC purity: 96% (tR 9.97 min). ESI-
MS: 474.33 [M].
2,6-Di([4-(3-methylphenyl)phenyl]ethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (40). Prepared from S34b and purified by
FCC (dichloromethane:methanol 30:1 to 20:1) to give the desired
product. Yield (18 mg) 78%. 1H NMR (400 MHz, CDCl3): δ 8.40 (t, J
= 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 4H), 7.65
(d, J = 8.0 Hz, 4H), 7.41−7.37 (m, 4H), 7.35 (t, J = 7.6 Hz, 2H), 7.22
(d, J = 7.2 Hz, 2H), 4.65 (s, 3H), 2.41 (s, 6H). 13C NMR (101 MHz,
CDCl3): δ 145.0, 143.9, 139.5, 138.9, 133.5, 129.4, 129.1, 128.1, 127.7,
124.4, 117.5, 109.0, 81.2, 45.5, 21.7 ppm. HPLC purity: 98.65% (tR
10.24 min). ESI-MS: 474.33 [M].
2,6-Di([4-(3-methoxyphenyl)phenyl]ethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (41). Prepared from S46 and purified by
FCC (dichloromethane:methanol 20:1 to 10:1) to give the desired
product. Yield (12 mg) 36%. 1H NMR (400 MHz, CDCl3): δ 8.39 (t, J
= 8.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 4H), 7.65
(d, J = 8.4 Hz, 4H), 7.37 (t, J = 7.6 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H),
7.11 (s, 2H), 6.94 (dd, J1 = 8.2 Hz, J2 = 2.0 Hz, 2H), 4.63 (s, 3H), 3.86
(s, 6H). 13C NMR (101 MHz, CDCl3): 160.3, 144.6, 143.9, 140.9,
139.3, 133.5, 130.8, 130.3, 127.7, 119.7, 117.7, 114.0, 113.0, 108.8,
81.3, 55.5, 45.5, 29.9 ppm. HPLC purity: 99% (tR 9.56 min). ESI-MS:
506.33 [M].
2,6-Di([4-(3-chlorophenyl)phenyl]ethynyl)-1-methylpyridinium
Trifluoromethanesulfonate (42). Prepared from S47 and purified by
FCC (dichloromethane:methanol 20:1 to 10:1) to give the desired
product. Yield (9 mg) 45%. 1H NMR (400 MHz, CDCl3): δ 8.43 (t, J
= 8.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.0 Hz, 4H), 7.65
(d, J = 8.0 Hz, 4H), 7.57 (s, 2H), 7.48 (d, J = 7.2 Hz, 2H), 7.42−7.37
(m, 4H), 4.70 (s, 3H). 13C NMR (101 MHz, CDCl3/MeOD): δ 143.8,
143.4, 141.0, 139.3, 134.9, 133.3, 130.7, 130.3, 128.5, 127.6, 127.1,
125.3, 117.9, 108.5, 80.8, 45.0, 29.5 ppm. HPLC purity: 99% (tR 10.20
min). ESI-MS: 514.33 [M].
Biology. Chemical and Reagents. [3H]Dofetilide (specific activity
82.3 Ci·mmol−1) was purchased from PerkinElmer (Groningen, The
Netherlands). Astemizole was purchased from Sigma-Aldrich (Zwijn-
drecht, The Netherlands). Bovine serum albumin (BSA, fraction V)
was purchased from Sigma (St. Louis, MO, USA). G418 was obtained
from Stratagene (Cedar Creek, USA). All the other chemicals were of
analytical grade and purchased from standard commercial sources.
Human embryonic kidney cells stably expressing the Kv11.1 channel
(HEK293 Kv11.1) were kindly provided by Dr Eckhard Ficker
(University of Cleveland, USA).
Cell Culture and Membrane Preparation. HEK293 Kv11.1 cells
were cultured and membranes were prepared and stored as described
previously.50
Radioligand Displacement Assay. The binding affinities of all
tested compounds was evaluated at 25 °C in a [3H]dofetilide
competitive displacement assay as reported earlier.2
Radioligand Competition Association Assay. The binding kinetics
of unlabeled compounds were determined at 25 °C using a
[3H]dofetilide competition association assay as described before,2
and the concentrations of unlabeled compounds used in this assay
were equivalent to their IC50 values.
Patch Clamp Assay. HEK293 Kv11.1 cells were cultured on 10 mm
glass coverslips and placed in a perfusion chamber (Cell Micro-
controls, Norfolk, USA) at room temperature and perfused with
K
J. Med. Chem. XXXX, XXX, XXX−XXX