Notes
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 17 3301
Deter m in a tion of log D7.4 Va lu es. Solutions of com-
pounds at a concentration of 100 µg/mL in methanol were
injected (20 µL) onto a Phase Sep Spherisorb C-8 column (3
µM, 15 cm × 4.6 mm) and eluted with varying percentages of
MeOH (60-90%, 3-4 concentrations/compound) in 15 mM
HEPES/0.15% triethylammonium phosphate buffer at pH 7.4
with a flow rate of 1 mL/min (at ambient temperature, with
detection at 220 nm). Values of log k′ were obtained from log
k′ ) log[(t - to)/to], where t is the retention time of the
compound and to is that of MeOH. Linear extrapolation of log
k′ to 0% MeOH yielded values of log kw.
Values of log D7.4 were also measured for representative
compounds using the “shake-flask” method (in octanol/15 mM
Hepes/135 mM NaCl at pH 7.4). Using the regression equation
log D7.4 ) 1.22 log kw - 0.51 (r ) 0.993) derived from these
data, log D7.4 values were then calculated from log kw for the
remaining para-substituted diphenylguanidines.
18 would therefore not be predicted on this basis, but
the presence of a hydroxyl group in this compound
would be expected to substantially increase H-bond
donation, which has been reported to reduce brain
uptake.26 Compound 12 is highly potent in vivo and
also highly lipophilic, suggesting that other mechanisms
may contribute to its high antiseizure activity. One
possibility is membrane activity, which is likely for
cationic compounds with long alkyl side chains. Changes
in membrane fluidity have been reported to be induced
by some anticonvulsant drugs.27-29 Additional param-
eters that could affect in vivo activity and may also be
dependent on lipophilicity include solubility and protein
binding in plasma, absorption, and clearance.
Biologica l Meth od s. Ver a tr id in e-In d u ced [14C]Gu a n i-
d in e F lu x Assa y: CNaIIA-I cells were obtained from the
Catterall Lab (University of Washington, Seattle, WA). The
cells were plated at 3 × 106 cells/96-well plate up to passage
20 and fed 24 h before use.
Con clu sion
A series of N,N′-diarylguanidines were synthesized
and studied for voltage-gated sodium channel blockade
in the guanidinium flux assay and anticonvulsant
activity in the audiogenic DBA/2 mouse model. Among
the substituents studied on the diphenylguanidines,
simple n-alkyl or n-alkoxy groups were favored over
phenyl or hydroxyalkyl groups. The more potent com-
pound in vitro that is also highly active in the in vivo
model is N,N′-bis(n-butylphenyl)guanidine (10) with an
IC50 of 0.13 µM in the guanidinium flux assay and 85%
inhibition of seizures at 25 mg/kg in the audiogenic
DBA/2 mouse model. All of the compounds of the series
showed only weak NMDA receptor ion channel activity.
It appears that this series of N,N′-diarylguanidines is
active in the audiogenic DBA/2 mouse model partly
through their blockade of neuronal sodium channels. An
investigation into the relationship of in vitro and in vivo
activity of this compound series with their measured/
calculated lipophilicities suggested that other physico-
Compounds were dissolved in DMSO and MeOH and diluted
for dose-response curves from 50 to 0.006 µM in preincubation
buffer (5.4 mM KCl, 0.8 mM MgSO4, 50 mM HEPES, 130 mM
choline chloride, 0.1 mg/mL BSA, 1.0 mM guanidine-HCl, and
5.5 mM D-glucose). Veratridine was dissolved at 400 mM in
MeOH and diluted in preincubation buffer (final assay con-
centration of 200 µM). [14C]Guanidine was also prepared in
preincubation buffer (final assay concentration 0.125 µCi/well).
Cultures are rinsed with and allowed to equilibrate in
preincubation buffer for at least 10 min before flux was
initiated with the addition of drugs, veratridine, and [14C]-
guanidine and incubated for 1 h at room temperature. Flux
was terminated by rinsing with ice-cold wash buffer (163 mM
choline chloride, 0.8 mM MgSO4, 1.8 mM CaCl2, 5.0 mM
HEPES, and 1.0 mg/mL BSA). The plates are then aspirated
dry, 100 µL of Optiphase “HiSafe” (Wallac) scintillation fluid
was added per well, and the plates were sealed and counted
on a Wallac Microbeta 1450 scintillation counter. IC50 values
were calculated using nonlinear regression.
Au d iogen ic DBA/2 Mou se Mod el: Testin g P r oced u r es
Mice (J ackson Labs, ME; weight range 6.5-12 g, 20-23 days
of age) were placed individually in a glass jar (25 cm i.d.) and
exposed to pure tone sound of 12 kHz and 120 dB for 45 s.
Animals were injected ip with the drug or vehicle (0.3 M
mannitol, in a volume of 10 mL/kg of body weight) 30 min prior
to exposure to the sound, unless otherwise noted. All experi-
ments were done in a fully blinded fashion and took place
between 11 a.m. and 5 p.m., and % response inhibition ) (MRS
control - MRS treatment)/MRS control × 100. Mean response
scores (MRS) were calculated as the average seizure score of
a test group of mice on our scale from 0 to 4. The J onckheere
nonparametric trend test, one-sided, was used to determine
the lowest effective dose in dose-response studies. Otherwise,
the Kruskal-Wallis nonparametric test was used, with Dunn’s
post-hoc test.
chemical parameters including electronic effects, pKa/b
,
may also be relevant for in vivo activity. This compound
series may generate agents of potential therapeutic
utility for a range of neurodegenerative disorders.
Exp er im en ta l Section
Ch em istr y. Melting points were determined in open
capillary tubes on a Thomas-Hoover apparatus and are uncor-
rected. Thin-layer chromatography was performed on Merck
silica gel 60 F254 (0.2 mm) or Baker-flex 1B2-F silica gel plates.
Guanidines were visualized on TLC with 254-nm UV light or
as a blue spot with bromcresol spray reagent (Sigma Chemical
Co.). Preparative TLC was performed on Analtech GF pre-
coated silica gel (1000 µM) glass-backed plates (20 × 20 cm).
The IR and NMR spectra of all compounds were consistent
with their assigned structures. NMR spectra were recorded
on a General Electric QE-300 spectrometer, and the chemical
shifts are reported in ppm (δ) relative to the residual signal
of the deuterated solvent (CHCl3, δ 7.26; CHD2OD, δ 3.30).
Infrared spectra were recorded in CHCl3 (unless otherwise
noted) on a Nicolet 5DXB FT-IR or Perkin-Elmer model 1420
spectrometer. All new compounds were analyzed either for
C, H, and N elemental analyses or for exact mass. Elemental
analyses were performed by either Desert Analytics (Tucson,
AZ) or Galbraith Laboratories (Knoxville, TN). High-resolu-
tion mass spectra (HRMS) were recorded on a Finnegan MAT
90 instrument. HPLC analyses were performed on a C18
reverse-phase column using 50:50 water/acetonitrile with 0.1%
TFA as the mobile phase.
Ack n ow led gm en t. Thanks to Sonia Connaughton
for carrying out some in vitro biological assays and
Robert N. McBurney for helpful discussions during the
preparation of this manuscript.
Refer en ces
(1) Goodman and Gilman’s The Pharmacological Basis of Thera-
peutics, 8th ed.; Gilman, A. G., Rall, T. W., Nies, A. S., Taylor,
P., Eds.; Pergamon Press: New York, 1991; p 312.
(2) Triggle, D. J .; Langs, D. A. Ligand Gated and Voltage-gated ion
Channels. Annu. Rep. Med. Chem. 1990, 25, 225-234.
(3) See ref 1, p 847.
(4) (a) See ref 1, p 440. (b) See ref 1, p 447.
(5) Squire, I. B.; Lees, K. R.; Pryse-Phillips, W.; Kertesz, A.;
Bamford, J . Efficacy and Tolerability of Lifarizine in Acute
Ischemic Stroke. A Pilot Study. Lifarizine Study Group. Ann.
N. Y. Acad. Sci. 1995, 765, 317-318.
The general experimental details for the synthesis of sym-
metrical and unsymmetrical N,N′-diarylguanidines have been
described previously.17