2586 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Benes et al.
1
185-188 °C (lit.31 mp 188-192 °C); H NMR (CDCl3) δ 7.7-
chloride, was applied in the eyes of all animals immediately
before placement of corneal electrodes. Abolition of the hindleg
tonic extensor component was used as the endpoint. These
experimental conditions produced tonic-clonic convulsions in
97% of animals tested, and only rats showing typical tonic-
clonic convulsions were used.22 All rats were submitted to a
maximum of 3 MES sessions: the first MES session was
performed to screen the animals and select those rats present-
ing a typical convulsive behavior. The day after, rats were
given the compounds to be tested or the vehicle and submitted
to a second MES session 2 or 4 h after the administration of
test compounds. The third MES session was performed at 6,
8, or 12 h after the administration of test compounds. The time
interval between each MES session was at least 4 h (rats
tested at 2 h were retested at 6 h and rats tested at 4 h were
retested at 8 h). The evaluation of the anticonvulsive profile
of test compounds was based on the duration of the tonic phase
(in seconds), each rat being its own control (internal control)
as obtained in the first MES session. An external control group
was also studied; in this particular case, rats were given the
vehicle and submitted to the three MES sessions procedure
as described above. All compounds used were suspended in
0.5% carboxymethylcellulose (4 mL/kg) and given by stomach
tube. In some experiments, compounds used were dissolved
in dimethyl sulfoxide (DMSO) (2 mL/kg) and given ip.
7.1 (m, 8H, Ar-H), 5.3, 4.7 (2 × br s, 1H, C10-H), 4.9 (br s,
2H, NH2), 3.7, 2.7 (2 × br s, 1H, -OH), 3.5 (m, 1H, C11-H),
3.0 (m, 1H, J ) 15 Hz, C11-H). Anal. (C15H14N2O2) C, H, N.
(R)-(-)-10,11-Dih yd r o-10-h yd r oxy-5H -d ib en z[b,f]a ze-
p in e-5-ca r boxa m id e (7). To a stirred solution of l-(-)-
menthoxyacetic acid chloride (16.5 g, 77 mmol) in dichlo-
romethane (160 mL) was added the racemic alcohol 8 (17.78
g, 70 mmol), followed by pyridine (8.13 mL, 100 mmol) and
4-(dimethylamino)pyridine (489 mg, 4 mmol), and the reaction
mixture was stirred for 16 h. After standard workup and
crystallization from dichloromethane/ethyl acetate to constant
melting point and optical rotation, 7.57 g (24%) of l-(-)-
menthoxyacetate was obtained as a white solid: mp 201-202
1
°C; H NMR (CDCl3) δ 7.6-7.15 (m, 8H, Ar-H), 6.5, 6.1 (2 ×
br s, 1H, C10-H), 4.8 (br s, 2H, NH2), 4.1 (m, 2H, -COCH2O-
), 3.6 (m, 1H, C11-H), 3.2 (m, 2H, C11-H, C1′-H), 2.3 (m, 1H,
C5′-H), 2.1 (m, 1H, C6′-H), 1.65 (m, 2H, C3′-H, C4′-H), 1.3 (m,
2H, C2′-H, C7′-H), 0.9 (m, 9H, -(CH3)2, C3′-H, C4′-H, C6′-H), 0.7
20
(m, 3H, CH3); [R]D ) 20.2° (c 0.51, pyridine); HPLC 98.5% of
(R)-enantiomer. Anal. (C15H14N2O2) C, H, N.
The menthoxyacetate was hydrolyzed at room temperature
by aqueous methanolic sodium hydroxide solution to the title
20
compound (3.88 g, 91%): mp 189-191 °C; [R]D ) -196° (c
20
0.87, ethanol) (lit.17 [R]D ) -197°, ethanol).
(S)-(+)-10,11-Dih yd r o-10-h yd r oxy-5H -d ib en z[b,f]a ze-
p in e-5-ca r boxa m id e (6). The mother liquors after prepara-
tion of the above diastereoisomer of menthoxyacetate were
hydrolyzed as described above, and the (S)-enantiomer-
enriched alcohol was resolved by D-(+)-menthoxyacetic acid
chloride. The menthoxyacetate (11.0 g, 35%), mp 201-202 °C,
2. Rota r od Test. Rats were examined for motor toxicity in
the rotating rod apparatus (Accelerator Rota-Rod (J ones &
Roberts) 7750; Ugo Basile). Naive rats were trained to hold
onto the 5-cm diameter neoprene rubberized cylinder until able
to maintain the equilibrium for 3 min while rotating at 6 rpm.
The day after, rats were given ip the test compound (dissolved
in DMSO, 2 mL/kg) and 15 min later were placed on the
rotating rod at a speed of 6 rpm. In a compound-treated rat
the neurological deficit is indicated by the inability of the rat
to maintain equilibrium for 1 min in each of three trials.32
20
[R]D ) -19.7° (c 0.54, pyridine), was hydrolyzed to the title
compound (5.77 g, 93%): mp 189-191 °C; [R]D20 ) 199° (c 1.10,
pyridine); HPLC 99.5% of (S)-enantiomer. Anal. (C15H14N2O2)
C, H, N.
(S)-(-)-10-Acetoxy-10,11-dih ydr o-5H-diben z[b,f]azepin e-
5-ca r boxa m id e (12). Gen er a l Meth od A: (S)-(+)-Alcohol 6
(25.4 g, 0.1 mol), pyridine (9.89 g, 0.125 mol), 4-(dimethyl-
amino)-pyridine (0.3 g, 2.46 mmol), and acetyl chloride (9.82
g, 0.125 mol) in dichloromethane (200 mL) were stirred at room
temperature for 1 h. Standard workup followed by crystal-
lization furnished (S)-12 (24.59 g, 83.5%): mp 186-187 °C;
3. Block a d e of Volta ge-Sen sitive Sod iu m Ch a n n els.
Blockade of voltage-sensitive sodium channels was studied by
investigating [3H]batrachotoxinin A 20-R-benzoate ([3H]BTX)
displacement binding to rat cortical synaptosomes. Rat cere-
bral cortical synaptosomes were prepared by differential
sucrose-Percoll density gradient centrifugations, as previously
described.33 Binding studies were performed by incubation of
[3H]BTX for 30 min at 37 °C with 42-680 µg of membrane
protein in a final volume of 200 µL in a solution containing
KCl (130 mM), MgSO4 (0.8 mM), glucose (5.5 mM), HEPES
(50 mM, pH 7.4), scorpion toxin (0.5 µM), tetrodotoxin (1 µM),
[3H]BTX (10 nM), bovine serum albumin (1 mg/mL), and
varying concentrations of competing compounds (0.1-1000
µM), as previously described.27 The binding reactions, per-
formed in 96-well EIA/RIA plates (Costar) with 300-µL capac-
ity, were initiated by the addition of 50 µL of the synaptosomal
solution to 150 µL of the reaction mixture. The specific binding
was calculated by subtraction of the nonspecific binding, which
was determined in the presence of 300 µM veratridine. The
binding reactions were stopped by vacuum filtration (Tomtec
Harvester 96) through glass fiber filtermats A (1450-21 from
Wallac) and washing of the filters and incubation tubes with
6 mL of an ice-cold wash solution consisting of choline chloride
(130 mM), CaCl2 (1.8 mM), MgSO4 (0.8 mM), bovine serum
albumin (1 mg/mL), and 5 mM HEPES/TRIS (pH 7.4). The
filtermats were dried, impregnated with MeltiLex A scintil-
lation mixture (Wallac), and inserted into plastic sample bags
(Wallac), and the radioactivity was counted in a 1450 Micro-
Beta spectrophotometric detector for 2 min with an efficiency
of 55-60%. [3H]BTX (specific activity: 50.50 Ci/mmol) was
from DuPont NEN. Tetrodotoxin, veratridine, and scorpion
toxin from Leiurus quinquestriatus hebraeus were from Sigma.
Scorpion toxin was resuspended in ice-cold distilled water (1
mg/mL) and incubated for 1 h at 0 °C. The mixture was then
centrifuged at 12000g for 10 min and the supernatant added
to the incubation medium. As previously reported,34 this
supernatant was composed by a major band of 7 kDa, spread-
ing from 4 to 14 kDa, upon FPLC separation (data not shown).
20
1
[R]D ) -20.5° (c 1.0, pyridine); H NMR (CDCl3) δ 7.6-7.1
(m, 8H, Ar-H), 6.4, 6.0 (2 × br s, 1H, C10-H), 5.0 (br s, 2H,
NH2), 3.6, 3.2 (2 × m, 2H, C11-H), 2.1 (s, 3H, -CH3); 13C NMR
(CDCl3) δ 171.1, 170.6 (CO), 157.4, 157.0 (NCON), 141.7, 141.0,
139.4, 134.8, 133.9 (quaternary C) 131.6, 131.4, 129.6, 129.2,
128.7, 128.6, 128.5, 128.2, (ArC), 72.7, 70.5 (C10), 36.4, 36.1
(C11), 21.6 (CH3); MS m/z 296 (M+), 193 (100); HPLC 99.8% of
(S)-enantiomer. Anal. (C17H16N2O3) C, H, N.
Ra cem ic 10,11-Dih yd r o-10-(n icotin oyloxy)-5H-d iben z-
[b,f]a zep in e-5-ca r boxa m id e (25). Gen er a l Meth od B: Ra-
cemic alcohol 8 (2.54 g, 10 mmol) was suspended in dichlo-
romethane (50 mL), and dicyclohexylcarbodiimide (2.27 g, 11
mmol) was added followed by nicotinic acid (1.35 g, 11 mmol)
and 4-(dimethylamino)pyridine (122 mg, 1 mmol). The reaction
mixture was stirred at room temperature overnight and
filtered, and after standard workup and chromatography using
stepwise gradient from dichloromethane to 2% methanol in
dichloromethane, the title compound 25 (2.74 g, 76%) was
obtained as white crystals: mp 195-196 °C (ethyl acetate/
ether); 1H NMR (CDCl3) δ 9.3, 9.1 (2 × br s, 1H, pyridine C2-
H), 8.8 (d, 1H, J ) 5 Hz, pyridine C6-H), 8.4, 8.2 (2 × br s,
1H, pyridine C4-H), 7.65-7.1 (m, 9H, pyridine C5-H and Ar-
H), 6.7, 6.3 (2 × br s, 1H, C10-H), 4.8 (br s, 2H, NH2), 3.8, 3.3
(2 × br s, 2H, C11-H). Anal. (C21H17N3O3) C, H, N.
P h a r m a cologica l Meth od s. All compounds were tested
using male Wistar rats (Harlan-Interfauna Ibe´rica, Barcelona,
Spain, or Gulbenkian Science Institute, Oeiras, Portugal)
ranging from 6 to 8 weeks old.
1. MES Test. MES stimulation was applied for 0.2 s, using
a Ugo Basile ECT unit 7801, with a frequency of 100 Hz, pulse
width of 0.6 ms, and current of 150 mA through bipolar corneal
electrodes. A drop of electrolyte/anesthetic, oxibuprocaine