A.-M. Liberatore et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3521–3523
3523
lation cocktail (Packard). Nonspecific binding (NSB) was
determined in the presence of 100 lM flunarizine, and the
total binding was obtained without inhibitor.
For each concentration of compound, percent of control is
calculated as follows:
bamazepine. In addition, the compounds tested behave
as functional Naþ channel blockers in a cellular assay.
Imidazoles 2 merit further study in terms of electro-
physiological use-dependency and in in vivo models.
ððcpmcompound ꢀ cpmNSBÞ ꢁ 100Þ=ðcpmcontrol ꢀ cpmNSBÞ:
Points are performed in duplicate.
Acknowledgements
For each compound the values included in the linear part of
each experiment’s sigmoid were retained in a linear regres-
sion analysis and were used to estimate the 50% inhibitory
concentration (IC50).
ꢀ
We would like to thank Jose Camara and his team for
mass spectral and NMR H analysis.
1
7. Pauwels, P. J.; Van Assouw, H. P.; Leysen, J. E.; Janssen,
P. A. Mol. pharmacol. 1989, 36, 525–531. Veratridine-
induced cytotoxicity on SHSY5Y cell line. SH-SY5Y
neuroblastoma cells were seeded on 96 well plates 24 h
prior to treatment. Cells were then preincubated with
compounds for 20 min before addition of 100 lM veratri-
dine and 1 mM ouabaine mixture. After 3 h exposure the
medium was replaced by a culture medium containing 10%
alamar blue and incubated for 18–24 h. Cytotoxicity was
evaluated after plate reading on a spectrophotometer at
double wave length (570, 620 nM). For each concentration
of compound the percentage of cell injury was calculated as
follows:
References and notes
1. Clare, J. J.; Tate, S. N.; Nobbs, M.; Romanos, M. A. Drug
Discovery Today 2000, 5, 506–520.
2. Thurieau, C. A.; Poitout, L. F.; Galcera, M.-O.; Moinet, C.
P.; Gordon, T. D.; Morgan, B. A.; Bigg, D. C.; Pommier, J.
WO 9964420, Chem. Abstr. 1999, 132, 35702.
3. Gordon, T. D.; Singh, J.; Hansen, P. E.; Morgan, B. A.
Tetrahedron Lett. 1993, 34, 1901.
4. Frechet, J. M. J.; Farall, M. J. J. Macromol. Sci., Chem.
1997, 507.
ðODvalues of veratridine treated cells ꢀ ODvalues of control cellsÞ=
5. Rather, J. B.; Rfid, E. Emmet J. Am. Chem. Soc. 1919, 41,
75–83.
6. McKinnon, A. C.; Wyatt, K. M.; McGivern, J. G.;
Sheridan, R. D.; Brown, C. M. Br. J. Pharmacol. 1995,
115, 1103–1109.
ðODvalues of veratridine untreated cells ꢀ ODvalues of control cellsÞ ꢂ 100
Each OD was initial OD)blank OD. The IC50 value was the
concentration, which decreased the % of cell injury by 50%
and was derived by extrapolation from graphs of dose–
response relations. Values used for calculations are the
mean values of triplicates.
Preparation of cortex membranes for Naþ binding: Cortex
membranes were prepared as follow. Rats were decapitated,
the brains rapidly removed and the cortices dissected and
weighed. The isolated cortex was homogenised by means of
a Teflon-glass homogeniser in 10 volumes of ice-cooled
0.32 M sucrose-5 mM potassium hydrogen phosphate
(pH 7.4, 4 °C.) solution. The resulting homogenate was
centrifuged at 1000g (4 °C) for 10 min and the supernatant
was further centrifuged at 20,000g (4 °C) for 15 min. The
pellet was suspended and washed in 0.32 M sucrose buffer
and centrifuged again at 20,000g (4 °C) for 15 min. The
residue was recovered. The membrane sample thus
obtained was suspended in Na-free assay buffer (50 mM
HEPES, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose,
130 mM choline chloride (pH 7.4) to give a final concen-
tration of about 4 mg protein/mL and stored at )80 °C until
use. Protein concentration was determined by the Bradford
method using bovine serum albumine as a standard.
Naþ binding protocol: Binding studies were carried out as
follow. 100 lL of the above membrane sample preparation
(75 lg/mL) was added to buffer containing 1 lM tetrodo-
toxin, 50 lg/mL scorpion venom, 5 nM [3H] BTX (34.0 Ci/
mmol) and the compound at different concentrations to
make a final volume of 0.5 mL. The reaction was carried
out at 25 °C and was terminated after 90 min by the
addition of 2 mL of ice-cold washing buffer (5 mM HEPES,
1.8 mM CaCl2, 0.8 mM MgSO4, 130 mM choline chloride,
(pH 7.4). The mixture was immediately vacuum filtered on
a unifilter GF/C (Packard) presoaked with 0.1% poly-
ethylene-imine. The filter was washed once with 2 mL of
ice-cold washing buffer. Bound [3H] BTX was determined
by liquid scintillation spectrometry in Microscint 0 scintil-
8. Selected data for compound 2g: Typical experimental
procedure. Synthesis of 2g: 4-(1,10-biphenyl-4-yl)-2-(1-
pentylhexyl)-1H-imidazole (2g). Caesium carbonate
(2.03 g, 6.25 mmol) was added to a solution of dipentylace-
tic acid (2.5 g, 12.5 mmol) in methanol (50 mL). The
reaction mixture was stirred for 1 h, the solvent was
evaporated, and 40-phenyl bromoacetophenone (3.43 g,
12.5 mmol) and dimethylformamide (40 mL) were added
and the mixture stirred overnight. The reaction mixture was
evaporated to dryness under reduced pressure and xylene
(60 mL) added to the residue. The caesium bromide salt was
filtered, ammonium acetate (20 g) added to the filtrate and
the reaction mixture refluxed for 1.5 h using a Dean–Stark
apparatus. After cooling, the reaction was diluted with ice
water and ethyl acetate (200 mL). The organic phase was
washed with a saturated solution of sodium bicarbonate
(2 ꢂ 100 mL) followed by brine (100 mL), dried over sodium
sulfate, filtered and evaporated to dryness under reduced
pressure. Purification was carried out on a silica gel column
(mobile phase: 5% methanol in dichloromethane) The
evaporated fractions were suspended in diethyl ether
(50 mL), filtered and rinsed with the same volume of ether
to afford 0.46 g (10%) of 2g as a light cream powder.
Melting point: 177–178 °C.
1H NMR (400 MHz, DMSO-d6, d): 11.84 (br s, 1H), 7.83–
7.33 (m, 10H), 2.70 (br s, 1H), 1.66–1.58 (m, 4H), 1.22–1.11
(m, 12H), 0.8–0.83 (m, 6H). Elemental analysis for
C26H24N2: Theoretical: C 83.37%, N 7.48%, H 9.15%.
Found: C 83.42%, N 7.64%, H 8.85%.
Liberatore, Anne-Marie
