Anticonvulsant activity and interactions with neuronal voltage- dependent sodium channel of analogues of ameltolide

Article abstract of DOI:10.1021/jm9608772

Fifteen compounds related to ameltolide (LY 201116) were studied for (i) anticonvulsant potential in the maximal electroshock-induced seizures (MES) and the subcutaneous pentylenetetrazol (sc Ptz)tests in mice and rats and (ii) interactions with neuronal voltage-dependent sodium channels. Compounds were chosen ranging in anticonvulsant activity in mice from very active to inactive. The active compounds were defined as those protecting 50% of the animals at doses between 10 and 50 μmol/kg and inactive compounds as those protecting 50% of the animals at doses greater than 1 mmol/kg. The series studied included three N-(2,6-dimethylphenyl)benzamides (compounds 1, 2 (ameltolide), and 3), three N-(2,2,6,6-tetramethyl)piperidinyl-4-benzamides (compounds 4, 5, 6), one phenylthiourea (compound 7), five N-(2,6- dimethylphenyl)phthalimides (compounds 8, 9, 10, 13, and 14), two N- phenylphthalimide derivatives (compounds 11 and 12), and one N-(2,2,6,6- tetramethyl)piperidinyl-4-phthalimide (compound 15). Phenytoin (PHT) was employed as the reference prototype antiepileptic drug. After inital screening in mice, compounds 1, 2, 3, 5, 8, 9, 10, 13, and 14 were selected for further testing in rats. Anticonvulsant ED₅₀s (effective doses in at least 50% of animals tested) of compounds in the MES test were determined in rats dosed orally and amounted to 52 (1), 135 (2), 284 (3), 231 (8), 131 (9), 25 (10), 369 (13), 354 (14), and 121 (PHT) μmol/kg, compound 5 presenting with an ED₅₀ value higher than 650 μmol/kg. In our hands, the apparent IC₅₀s (inhibitory concentrations 50) of compounds toward binding to rat brain synaptosomes of [³H]batrachotoxinin-A-20α-benzoate were 0.25 (1), 0.97 (2), 0.35 .(3), 25.8 (5), 161.3 (8), 183.5 (9), 0.11 (10), 1.86 (13), 47.8 (14), and 0.86 (PHT) μM. The relationship between the activity in the MES test and the capacity to interact in vitro with neuronal voltage- dependent sodium channels and the fact that the IC₅₀ values obtained in the in vitro test are close to the brain concentrations at which anticonvulsant activities are reported to occur for ameltolide strongly suggest that the anticonvulsant properties of most compounds tested could be a direct result of their interaction with the neuronal voltage-dependent sodium channel.

Full text of DOI:10.1021/jm9608772

J . Med. Chem. 1998, 41, 3307-3313
3307
Articles
An ticon vu lsa n t Activity a n d In ter a ction s w ith Neu r on a l Volta ge-Dep en d en t
Sod iu m Ch a n n el of An a logu es of Am eltolid e
J oseph Vamecq,*,† Didier Lambert,‡ J acques H. Poupaert,‡ Bernard Masereel,§ and J ames P. Stables|
INSERM/ CHRU Lille, Domaine du Certia, 369 rue J ules Guesde, BP 39, 59651 Villeneuve d’Ascq Cedex, France,
Unite´ de Chimie The´rapeutique, Ecole de Pharmacie, Universite´ Catholique de Louvain, Tour Van Helmont,
Avenue E. Mounier 73, UCL 7340 B-1200 Brussels, Belgium, De´partement de Chimie The´rapeutique, Universite´ de Lie`ge,
Institut Gilkinet, Rue Fusch 3, B-4000 Lie`ge, Belgium, and Preclinical Pharmacological Section, Epilepsy Branch,
DCDND, NINDS, National Institutes of Health, Bethesda, Maryland 20892-9020
Received December 27, 1996
Fifteen compounds related to ameltolide (LY 201116) were studied for (i) anticonvulsant
potential in the maximal electroshock-induced seizures (MES) and the subcutaneous pentyl-
enetetrazol (sc Ptz) tests in mice and rats and (ii) interactions with neuronal voltage-dependent
sodium channels. Compounds were chosen ranging in anticonvulsant activity in mice from
very active to inactive. The active compounds were defined as those protecting 50% of the
animals at doses between 10 and 50 µmol/kg and inactive compounds as those protecting 50%
of the animals at doses greater than 1 mmol/kg. The series studied included three N-(2,6-
dimethylphenyl)benzamides (compounds 1, 2 (ameltolide), and 3), three N-(2,2,6,6-tetramethyl)-
piperidinyl-4-benzamides (compounds 4, 5, 6), one phenylthiourea (compound 7), five N-(2,6-
dimethylphenyl)phthalimides (compounds 8, 9, 10, 13, and 14), two N-phenylphthalimide
derivatives (compounds 11 and 12), and one N-(2,2,6,6-tetramethyl)piperidinyl-4-phthalimide
(compound 15). Phenytoin (PHT) was employed as the reference prototype antiepileptic drug.
After inital screening in mice, compounds 1, 2, 3, 5, 8, 9, 10, 13, and 14 were selected for
further testing in rats. Anticonvulsant ED50s (effective doses in at least 50% of animals tested)
of compounds in the MES test were determined in rats dosed orally and amounted to 52 (1),
135 (2), 284 (3), 231 (8), 131 (9), 25 (10), 369 (13), 354 (14), and 121 (PHT) µmol/kg, compound
5 presenting with an ED50 value higher than 650 µmol/kg. In our hands, the apparent IC50s
(inhibitory concentrations 50) of compounds toward binding to rat brain synaptosomes of
[3H]batrachotoxinin-A-20R-benzoate were 0.25 (1), 0.97 (2), 0.35 (3), 25.8 (5), 161.3 (8), 183.5
(9), 0.11 (10), 1.86 (13), 47.8 (14), and 0.86 (PHT) µM. The relationship between the activity
in the MES test and the capacity to interact in vitro with neuronal voltage-dependent sodium
channels and the fact that the IC50 values obtained in the in vitro test are close to the brain
concentrations at which anticonvulsant activities are reported to occur for ameltolide strongly
suggest that the anticonvulsant properties of most compounds tested could be a direct result
of their interaction with the neuronal voltage-dependent sodium channel.
In tr od u ction
and, most recently, topiramate,8 are also employed.
Further, a number of AEDs (i.e., tiagabine,8 losiga-
mone,9 remacemide,10 D-23129,11 and oxcarbazepine8)
are now in human clinical experimentation. In recent
years, the field of AED development is quite dynamic,
affording many promising research opportunities.
More than half of a century has elapsed since the
anticonvulsant properties of phenytoin were first evi-
denced in laboratory animal models1 with successful
therapeutic administration in epileptic patients.2,3
Phenytoin is still one of the most commonly used
antiepileptic drugs (AEDs). Today, in addition to
phenytoin, several AEDs are widely utilized in the
treatment of the various forms of epilepsy.3,4 Some of
the other standard drugs include carbamazepine, phe-
nobarbital, ethosuximide, valproic acid, and various
benzodiazepines. In addition, several newer agents
γ-vinyl-GABA,4 felbamate,5 gabapentin6, lamotrigine,7
Our work is based on the drug ameltolide, a potent
AED previously investigated by Eli Lilly, having orig-
inally emerged from the laboratories of Clark and co-
workers.12-18 This group of investigators isolated the
promising 4-aminobenzamide pharmacophore which
subsequently led to the fruitful design of several new
and potent anticonvulsant compounds. Based on po-
tency-structure relationships, we designed analogues
related to ameltolide and phenytoin. This resulted in
* Corresponding author. Fax: (+33) 3 20 05 91 72.
† INSERM/CHRU Lille.
‡ Universite´ Catholique de Louvain.
§ Universite´ de Lie`ge.
providing
a family of N-phenylphthalimide com-
| National Institutes of Health.
pounds.19-22 Preliminary studies pointed out that the
S0022-2623(96)00877-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/11/1998

3308 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Vamecq et al.
Ta ble 1. Anticonvulsant and Neurotoxicity Screening Data in
Mice Dosed Intraperitoneally with Ameltolide and Related
Compoundsa
MES test
sc Ptz test
toxicity
30 min
compd
30 min
4 h
30 min
4 h
4 h
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
+++
+++
+++
-
++
++
++
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
+
-
-
-
+
-
-
-
-
++
-
-
-
+
-
-
-
-
-
+
-
-
-
+
++
-
-
-
-
-
-
+++
-
+
+++
-
-
-
-
-
+++
-
-
++
++
+++
-
-
++
++
-
-
+++
-
-
+
-
++
++
-
++
++
-
-
-
-
a
The anticonvulsant (MES and sc Ptz tests) and neurotoxicity
activities were determined 30 min and 4 h after the administration
of compounds. The symbols +++, ++, and + signify activity at
30, 100, and 300 mg/kg, respectively; - denotes no activity
observed at 300 mg/kg. Toxicity was determined by the rotorod
test. Abbreviations: MES, maximal electroshock-induced seizures;
sc Ptz, subcutaneous pentylenetetrazol.
F igu r e 1. Compounds 1-15.
N-phenylphthalimides exhibit anticonvulsant activity16-19
similar to the profile of phenytoin, a prototype AED
shown to interact with neuronal voltage-dependent
sodium channels.23,24
offered in the MES test as defined above) anticonvulsant
molecules. This large range of MES anticonvulsant
activity was chosen with the view that the collection of
a wide range of responses in the synaptosomal votage-
dependent sodium channel binding test would aid in
reaching conclusive SAR evaluations.
In this paper, we report the anticonvulsant and
neurotoxic properties of 15 compounds structurally
related to ameltolide and their ability to interact with
the voltage-dependent sodium channel from rat brain
synaptosomal fractions as evidenced by inhibition of
[3H]batrachotoxinin-A-20R-benzoate binding. Com-
pounds could not be chosen on the basis of SAR that
would be discovered afterward as a result of [3H]batra-
chotoxinin-A-20R-benzoate binding test data. Com-
pounds were designed and synthesized after classical
medicinal chemistry practices and were chosen on the
basis of their level of anticonvulsant activity ranging
from very active to moderately active and to inactive,
i.e., with activity in the MES tests at 30, 100, and 300
mg/kg, respectively. We investigated whether a cor-
relation could be obtained between interactions with the
neuronal voltage-dependent sodium channel and activi-
ties in the maximal electroshock-induced seizures (MES)
test. The view that the in vivo anticonvulsant activity
of ameltolide (and several related compounds) is largely
mediated by its interaction with the neuronal voltage-
dependent sodium channel is supported by the present
experimentation.
Resu lts
An ticon vu lsa n t a n d Neu r otoxicity Scr een in g
Da ta in Mice Dosed In tr a p er iton ea lly (ip ). Initial
ip screening for anticonvulsant activity and toxicity in
mice is documented in Table 1. Compounds 1, 2, and 3
showed activity in the MES test at 30 mg/kg. Com-
pound 3 further exhibited activity in the subcutaneous
pentylenetetrazol (sc Ptz) seizure test. Compounds 2
and 3 did not present with toxicity, whereas compound
1 was toxic at 300 mg/kg. Compounds 4 and 6 were
devoid of both anticonvulsant activity and neurotoxicity.
Compound 5 was active at 30 mg/kg in the MES but
not in the sc Ptz seizure test, showing toxicity, however,
at 300 mg/kg. Compound 7 was also active in the MES
test without displaying activity in the sc Ptz seizure test
or toxicity at the doses studied. Compound 8 was active
only in the MES test at 100 mg/kg and did not exhibit
toxicity at 300 mg/kg. Compounds 9 and 10 were active
in the MES (at 100 and 30 mg/kg, respectively) and in
the sc Ptz test (at 300 and 30 mg/kg, respectively), and
exhibited toxicity at 300 and 100 mg/kg, respectively.
Compounds 11 and 12 were inactive in the sc Ptz test
and not toxic, the latter being active only at high doses
(300 mg/kg) in the MES test. Compounds 13 and 14
were active in the MES test at 100 mg/kg but were
inactive at all doses in the sc Ptz test, the latter
compound being toxic at 300 mg/kg. Compound 15
failed to demonstrate any anticonvulsant protection or
neurotoxicity at the various doses studied.
Ch em istr y
The structures of 15 compounds related to ameltolide
(compound 2) are shown in Figure 1. The series
includes three N-(2,6-dimethylphenyl)benzamides (com-
pounds 1, 2, and 3), three N-(2,2,6,6-tetramethyl)-
piperidinyl-4-benzamides (compounds 4, 5, and 6), one
phenylthiourea (compound 7), five N-(2,6-dimethyl-
phenyl)phthalimides (compounds 8, 9, 10, 13, and 14),
as well as two other N-phenylphthalimide derivatives
(compounds 11 and 12), and one N-(2,2,6,6-tetramethyl)-
piperidinyl-4-phthalimide (compound 15). Compounds
were synthesized by standard methods. They included
very active, moderately active, and inactive (protection
An ticon vu lsan t (an ti-MES) Scr een in g an d Qu an -
tita tive Eva lu a tion Da ta in Ra ts Dosed Or a lly.
From the initial ip mouse screening, compounds 1, 2,
3, 5, 8, 9, 10, 13, and 14 were selected for further oral

Ameltolide Analogues and Sodium Channels
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3309
Ta ble 2. Anticonvulsant (anti-MES) and Toxicity Screening
Ta ble 3. Quantitative Evaluations of Selected Ameltolide
Analogues in MES Tests and Batrachotoxin-Binding
Experiments Performed in Ratsa
Data in Rats Dosed Orally with Ameltolide Analoguesa
compd 15 min 30 min
1 h
2 h
4 h
toxicity
anticonvulsant ED50
apparent IC50
(10-6 M)
1
2
3
5
8
++++ ++++ ++++ ++++ ++++
++++ ++++ ++++ ++++ ++++
-
compd
(µmol/kg)
-
-
+++
-
++
-
+
+
-
1
2
3
5
8
9
52.3 ( 2.0
135 ( 3
284 ( 10
>650
231 ( 7
131 ( 7
25.3 ( 4.1
369 ( 14
354 ( 10
121 ( 7
0.25 ( 0.05
0.97 ( 0.14
0.35 ( 0.06
25.8 ( 5.0
161 ( 14
-
-
-
-
+++
+++
+++
+++
-
++
-
+++
++++
-
9
++++
-
10
13
14
++++ ++++ ++++ ++++ ++
-
-
-
-
-
+
-
-
+
+
+ (4 h)
184 ( 20
++
-
10
13
14
PHT
0.11 ( 0.03
1.86 ( 0.13
47.8 ( 7.3
0.86 ( 0.07
a
Rats were given a single dose of 30 mg compound/kg body
weight and anticonvulsant activities determined in the maximal
electroshock seizures (MES) test. No toxicity was observed at this
dose except for compound 13 at the time point 4 h. Symbols are
++++, activity in 75-100% of administered animals; +++, in
50-75% of animals; ++, in 25-50% animals; +, 0-25% animals;
and -, no activity or toxicity. At least four animals were utilized
for each time point.
a
Experimental measurements were made in quadriplicate and
results are mean values ( SD. Anticonvulsant ED50 values were
determined in the MES test performed in rats dosed orally.
Apparent IC50 were determined in the [3H]batrachotoxinin-A-20-
R-benzoate-binding test performed on rat brain synaptosomes.
PHT: phenytoin.
evaluation in rats. Anti-MES activities determined in
rats treated with 30 mg/kg of the selected compounds
are summarized in Table 2. No toxicity was observed
at this dose for any of the compounds, except for
compound 13 which showed toxicity in one of four
animals tested. Compounds 1, 2, and 10 showed the
best anticonvulsant activity. All animals dosed with
these compounds showed protection at 5 time points
from 15 min to 4 h. Intermediate anticonvulsant
protection was offered by compounds 8 and 9. Com-
pound 3 presented with moderate activity, giving,
however, a relatively good protection at the 30 min time
point. No anticonvulsant protection was induced by
compound 5, whereas administration of compounds 13
and 14 led to a relatively weak anticonvulsant protec-
tion.
These screening data were confirmed by quantitative
anticonvulsant evaluation of compounds in the MES test
performed in rats dosed orally. Doses of compounds
offering protection in 50% of test animals (i.e., ED50s)
were determined and are presented in Table 3. Mean
ED50 values amounted to (in order of increasing values
and decreasing efficacy of compounds) 25.3 (10), 52.3
(1), 120.7 (PHT), 130.6 (9), 135.2 (2), 230.8 (8), 284.1
(3), 354.0 (14), 369.0 (13), and more than 650 (5) µmol/
Kg.
Qu a n t it a t ive E va lu a t ion on Neu r on a l Sod iu m
Ch a n n els. The competitions of selected compounds
with the binding of [3H]batrachotoxinin-A-20R-benzoate
to the voltage-dependent sodium channels from rat
brain synaptosomes were studied and compared with
the displacement properties of phenytoin tested in the
same experimental model. Apparent IC50s offered in
this binding test by the compounds are presented in
Table 3. The mean apparent IC50 values were (in order
of increasing values and decreasing efficacy of com-
pounds) 0.14 (10), 0.25 (1), 0.35 (3), 0.86 (PHT), 0.97
(2), 1.86 (13), 25.8 (5), 47.8 (14), 161.3 (8), and 183.5
(9) µmol/L.
dependent sodium channels involves a site other than
the [3H]batrachotoxinin-A-20R-benzoate binding site29
(structure and function studies of voltage-sensitive ion
channels have been previously reviewed by Catterall30)
but presenting with allosteric modulating properties
toward the latter site.24 The present work reports on
the in vitro inhibition by 15 analogues related to
ameltolide of batrachotoxin binding to the neuronal
voltage-dependent sodium channel and the in vivo
anticonvulsant activity of compounds in the MES test.
Phenytoin (PHT) was utilized as a standard for which
anti-MES activity was accounted for by interaction with
Na channels. Anticonvulsant ED50 and apparent IC50
values of compounds may be linked through a semi-
logarithmic relationship involving half of the test mol-
ecules, i.e., PHT, compounds 1, 2, 10, and 14, and the
relationship appears to fit the following equation:
IC50
ED50 ) 130 log
9
where ED50 is expressed as µmol/kg and (apparent) IC50
as 10-8 M values (Figure 2). Compared to this basal
group of compounds (for which anti-MES activity may
be considered as resulting from inhibition of sodium
channels), compounds 3, 13, and 5, on one hand, and
compounds 8 and 9, on the other hand, present with
anticonvulsant ED50 values which are higher and lower,
respectively, than would be expected on the basis of
their ability to interact with Na channels (Figure 2). The
relatively lower anticonvulsant potencies of compounds
3, 13, and 5 might be explained by low bioavailability
of the compounds. The relatively higher anticonvulsant
potencies of compounds 8 and 9 may be a result of
relatively high bioavailability, mechanisms other than
only their inhibition of sodium channels or their delivery
as prodrug compounds. In this respect, 9 might give
rise in vivo to 2, and 8 to 13, through oxidation of the
nitro group to an amine and hydroxylation at the
position 4 of the phthalimide pharmacophore, respec-
tively. Plotting the EC50 and apparent IC50 data ac-
cording to a linear instead of a semilogarithmic rela-
tionship does not change the general conclusions of our
work, except that 13 instead of 14 should be considered
in the basal group including 1, 2 (ameltolide), 10, and
Discu ssion
Interactions of anticonvulsant agents with the neu-
ronal voltage-dependent sodium channel have been
previously documented for phenytoin, carbamazepine,23,25
ralitoline,26 lamotrigine,27 and U-54494A and metabo-
lites.28 Interaction of phenytoin with neuronal voltage-

3310 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Vamecq et al.
F igu r e 2. Relationships between anticonvulsant MES ED50 and batrachotoxin apparent IC50 values. Semilogarithmic plotting
of data from Table 3 is illustrated. Other comments are in the text (Discussion). PHT: phenytoin.
were obtained from Merck (Darmstadt, Germany). The [3H]-
PHT. In this view, at least on the basis of oral dosing,
batrachotoxinin-A-20R-benzoate was obtained from Amersham
14 would be considered more like 8 and 9, and only
compound 3 would present with anticonvulsant potency
less than expected on the basis of observed activity in
the BTX-binding test. It should be stressed that in mice
dosed intraperitonally, 3 was highly active in the MES
test and was endowed with an ED50 value of 24.8 µmol/
kg versus 31.8, 10.8, 49.5, and 37.5 µmol/kg for com-
pounds 1, 2 (ameltolide), 10, and PHT, respectively
(data not shown). The low level of activity recorded for
3 in the oral rat MES test might be related to a low
oral bioavailability or to rapid detoxifying conjugation
in this animal species.
Our data supports the view that the antiepileptic
activity of ameltolide largely documented in the litera-
ture13,14,16,18,31-35 is the result of its interaction with the
neuronal voltage-dependent sodium channel. A major
element in favor of this view is given by the IC50 we
recorded for ameltolide (0.9 µM), a concentration that
is commonly observed in the brain of animals receiving
this anticonvulsant agent. Potts et al.32 reported in rats
dosed orally with ameltolide (50 mg/kg) brain levels
amounting to 5-7 µg/g tissue (approximated to 20 µM)
2 h after dosing and 2 µg/g tissue (approximated to 6
µM) 5 h after administration. Tested in the same
experimental conditions, ameltolide (2) appears to be
as potent as phenytoin (PHT) in interacting with the
Na channels. The phthalimide (10) and nitro (1)
counterparts of ameltolide are several times more potent
than ameltolide and phenytoin in the binding test and
oral MES test in rats. For this reason, we believe that
these agents may represent promising new leads as
potential anticonvulsants.
International plc, Buckinghamshire, England.
An a lytica l P r oced u r es. Melting points (uncorrected)
were determined in open capillary tubes on a Totolli apparatus.
IR spectra were recorded on KBr disks using a Perkin-Elmer
IR spectrophotometer (Grating infrared spectrophotometer,
model 457). NMR spectra were measured on compounds in
solution in DMSO-d6 (or in CD3OD) with tetramethylsilane
as internal standard, at 300 MHz for 1H and 75 MHz for 13C,
on a BRUCHER AM 300 apparatus. HPLC analyses of
compounds were performed on a Lichrocart 125-R Licrospher
RP 18 column with a flow rate of 1 mL/min of a methanol-
water (65/35, v/v) mixture as elution solvent and with detection
at 260 nm. Elemental analyses (C, H, N) were performed on
a Carlo-Erba EA 1108 elemental analyzer. All compounds had
1
IR and H and 13C NMR spectra consistent with their assigned
structure. They were homogeneous in HPLC. Their micro-
analytical data were within ( 0.4% of the calculated figures.
Ch em istr y. 4-Nitr o-N-(2,6-d im eth ylp h en yl)ben za m id e
(1). 4-Nitrobenzoyl chloride (5 g, 26.9 mmol) was added
portionwise to 2,6-dimethylaniline (4.8 g, 40.4 mmol) dissolved
in a mixture of dry THF (50 mL) and dry pyridine (5 mL).
The reaction mixture was stirred for 3 h at room temperature
and then quenched with 500 mL of cold 1 N HCl. The
resulting precipitate was collected on a filter, washed with
water, and recrystallized from absolute ethanol (5.60 g, 78%
yield): mp 183-185 °C; 1H NMR (DMSO-d6) δ 2.22 (s, 6H,
CH3), 7.11-8.40 (m, 7H, aromatic), 10.15 (s, 1H, NH); 13C NMR
δ 18.29 (CH3), 123.72-149.62 (aromatic C), 164.29 (CdO); IR
(cm-1) 3300, 1650 (amide), 1600 (phenyl), 1500-1300 (nitro).
4-Am in o-N-(2,6-d im eth ylp h en yl)ben za m id e (2). 4-Ni-
tro-N-(2,6-dimethylphenyl)benzamide (1) (2 g, 8.13 mmol) was
dissolved in cyclohexene (150 mL), and then 10% palladium
on charcoal (250 mg) and 2-propanol (50 mL) were added. The
mixture was heated under reflux for 8 h and then filtered. The
solvent was evaporated in vacuo, and the residue was recrys-
tallized from an ethanol-water (1/1, v:v) mixture (1.77 g, 91%
yield). mp 210-212 °C. 1H NMR (DMSO-d6) δ 2.15 (s, 6H,
CH3), 6.59-7.76 (m, 7H, aromatic), 10.15 (s, 1H, NH); 13C NMR
δ 18.32 (CH3), 112.84-132.01 (aromatic C), 165.81 (CdO); IR
(cm-1) 3450 (amino), 3300, 1650 (amide), 1600 (phenyl).
Exp er im en ta l Section
Ma ter ia ls. Reagents were purchased from Sigma-Aldrich
(Saint-Quentin Fallavier, France; Bornem, Belgium). Solvents

Ameltolide Analogues and Sodium Channels
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3311
4-H yd r oxy-N-(2,6-d im et h ylp h en yl)b en za m id e
(3).
phthalic anhydride (5.5 g, 28.3 mmol), compound 9 was
synthesized in the same way as 8 and was recrystallized from
methanol-water (1/1, v:v) (6.65 g, 79% yield): mp 176-179
°C; 1H NMR (DMSO-d6) δ 2.10 (s, 6H, CH3), 7.24-8.73 (m,
6H, aromatic); 13C NMR δ 17.48 (CH3), 30.64 (CH3), 118.63-
151.70 (aromatic C), 164.29 (CdO); IR (cm-1) 1730-1680
(imide), 1600 (phenyl), 1350 (nitro).
4-Amino-N-(2,6-dimethylphenyl)benzamide (2) (0.35 g, 1.46
mol) was dissolved in a water-sulfuric acid solution (75/25,
v:v) (20 mL). The resulting solution was cooled to 0-5 °C. A
cold solution of NaNO2 (0.18 g, 2.6 mmol in 0.4 mL of water)
was added dropwise. The supernatant was added to a boiling
sulfuric acid solution (1.65 mL of sulfuric acid and 1.5 mL of
water). The reaction mixture was refluxed for 30 min. The
resulting mixture was poured onto ice and stirred to give a
precipitate which was filtered, washed with water, dried, and
recrystallized from a hydrochloric acid-water solution (1/1,
v:v) to give pure 3 (0.3 g, 75%): mp 233-235 °C; 1H NMR
(DMSO-d6) δ 2.14 (s, 6H, CH3), 6.63-7.08 (m, 7H, aromatic),
9.48 (s, 1H, NH); 13C NMR δ 18.25 (CH3), 115.07-135.80
(aromatic C), 164.81 (CdO); IR (cm-1) 3350 (amino), 3250
(hydroxyl), 1650 (amide), 1620 (phenyl).
4-Am in o-N-(2,6-d im eth ylp h en yl)p h th a lim id e (10). Us-
ing 4-nitro-N-(2,6-dimethylphenyl)phthalimide (9) (3 g, 10.1
mmol), compound 10 was synthesized in the same way as 2
and was recrystallized from ethanol-water (1/1, v:v) (2.02 g,
75% yield): mp 195-196 °C; 1H NMR (DMSO-d6) δ 2.10 (s,
6H, CH3), 7.18-7.64 (m, 6H, aromatic); 13C NMR δ 18.32 (CH3),
112.84-132.02 (aromatic C), 165.81, 165.02 (CdO); IR (cm-1
3300 (amino), 1620 (phenyl).
)
N-(3-Nitr o-2,6-d im eth ylp h en yl)p h th a lim id e (11). Us-
ing 3-nitro-2,6-dimethylaniline (4.68 g, 28 mmol) and phthalic
anhydride (5.5 g, 28 mmol), compound 11 was synthesized in
the same way as 8 and was recrystallized from methanol-
water (1/1, v:v) (6.40 g, 77% yield): mp 167-169 °C; 1H NMR
(DMSO-d6) δ 2.29-2.32 (s, 6H, CH3), 7.63-8.16 (m, 6H,
aromatic); 13C NMR δ 14.28 (CH3, (p-NO2)), 18.07 (CH3, (o-
NO2)),124.04-148.61 (aromatic C); IR (cm-1) 1780-1730
(imide), 1600 (phenyl), 1500-1300 (nitro).
N-[4-(2,2,6,6-Tetr am eth yl)piper idin -1-yl]ben zam ide (4).
Using benzoyl chloride (5 g, 35.5 mmol) and 4-amino-(2,2,6,6-
tetramethyl)piperidine (5 g, 31.99 mmol), compound 4 was
synthesized in the same way as 1. Compound 4 was recrystal-
lized from acetone/ether (1/1, v:v) (6.91 g, 83% yield): mp 227-
1
229 °C; H NMR (CD3OD-d6) δ 1.56 (s, 6H, CH3), 1.62 (s, 6H,
CH3), 1.80-2.17 (m, 5H, CH(CH2)2), 7.44-7.70 (m, 5H, aro-
matic), 9.50 (s, 1H, NH); 13C NMR δ 25.14 (CH3), 30.64 (CH3),
41.78-58.81 (piperidine C), 128.42-135.41 (aromatic C),
169.81 (CdO); IR (cm-1) 3440 (piperidine), 3400, 1650 (amide),
1610 (phenyl).
N-(3-Am in o-2,6-d im eth ylp h en yl)p h th a lim id e (12). Us-
ing N-(3-nitro-2,6-dimethylphenyl)phthalimide (11) (3 g,10.1
mmol), compound 12 was synthesized in the same way as 2
and was recrystallized from ethanol-water (1/1, v:v) (2.23 g,
83% yield): mp 165-166 °C; 1H NMR (DMSO-d6) δ 2.09 (s,
4-Nitr o-N-[4-(2,2,6,6-tetr a m eth yl)p ip er id in -1-yl]ben z-
a m id e (5). Using 4-nitrobenzoyl chloride (5 g, 26.9 mmol) and
4-amino-(2,2,6,6-tetramethyl)piperidine (2.41 g, 15.42 mmol),
compound 5 was synthesized in the same way as compounds
1 and 4. Compound 5 was recrystallized from acetone/ether
(1/1, v:v) (3.76 g, 80% yield): mp 262-264 °C; 1H NMR
(CD3OD-d6) δ 1.53 (s, 12H, CH3), 1.77-2.10 (m, 5H, CH(CH2)2),
8.14-8.34 (m, 4H, aromatic), 9.03 (s, 1H, NH); 13C NMR δ
24.14 (CH3), 30.64 (CH3), 40.59-58.74 (piperidine C), 124.51-
151.05 (aromatic C), 167.53 (CdO); IR (cm-1) 3500 (piperidine),
3300, 1650 (amide), 1600 (phenyl), 1500-1300 (nitro).
3H, CH3), 2.19 (s, 3H, CH3), 7.53-8.06 (m, 6H, aromatic); 13
C
NMR δ 14.10 (CH3, (p-NO2)), 18.07 (CH3, (o-NO2)), 124.04-
148.61 (aromatic C); IR (cm-1) 3300 (amino), 1780-1730
(imide), 1600 (phenyl).
4-Hydr oxy-N-(2,6-dim eth ylph en yl)ph th alim ide (13). Us-
ing 4-amino-N-(2,6-dimethylphenyl)phthalimide (10), com-
pound 13 was synthesized in the same way as 3 and was
recrystallized from a hydrochloric acid-water solution (1/1,
1
v:v) giving pure 13 (1.29 g, 97% yield): mp 170-172 °C; H
4-Am in o-N-[4-(2,2,6,6-tetr a m eth yl)p ip er id in -1-yl]ben z-
a m id e (6). Using 4-nitro-N-[4-(2,2,6,6-tetramethyl)piperidin-
1-yl]benzamide (5) (3 g, 9.81 mmol), compound 6 was synthe-
sized in the same way as 2 and was recrystallized from
ethanol-water (1/1, v:v) (1.60 g, 73% yield): mp 234-236 °C;
1H NMR (CD3OD-d6) δ 1.46 (s, 6H, CH3), 1.55 (s, 6H, CH3),
1.60-2.10 (m, 5H, CH(CH2)2), 6.68-7.70 (m, 4H, aromatic),
9.03 (s, 1H, NH); 13C NMR δ 15.86 (CH3), 30.64 (CH3), 41.78-
NMR (DMSO-d6) δ 2.10 (s, 6H, CH3), 7.24-8.74 (m, 6H,
aromatic); 13C NMR δ 17.49 (CH3), 118.65-151.74 (aromatic
C), 164.82, 165.09 (CdO); IR (cm-1) 3100 (hydroxyl), 1780-
1740 (imide), 1620 (phenyl).
4-Ch lor o-N-(2,6-d im et h ylp h en yl)p h t h a lim id e
(14).
4-Amino-N-(2,6-dimethylphenyl)phthalimide (10) (0.1 g, 0.45
mmol) was dissolved in a water-sulfuric acid solution (1/1,
v:v) (10 mL). The temperature was lowered to 0-5 °C, and
the mixture was stirred for 15 min. Cold NaNO2 solution (0.24
g in 1 mL water) was added dropwise at 0-5 °C. The
diazonium solution was then poured slowly in the cold solution
of CuCl. When the temperature reached 15 °C, the reaction
mixture was heated to 60 °C. The mixture was then filtered
and washed with 0.1 N aqueous solution of NaHCO3, with
distilled water, and finally with a 0.1 N aqueous solution of
H3PO4. Compound 14 was recrystallized from ethanol-water
(9/1, v:v) (0.083 g, 65% yield): mp 192-194 °C; 1H NMR
(DMSO-d6) δ 2.10 (s, 6H, CH3), 7.24-8.81 (m, 6H, aromatic);
13C NMR δ 17.63 (CH3), 118.79-151.87 (aromatic C), 164.96,
165.22 (CdO); IR (cm-1) 1730-1680 (imide), 1625 (phenyl),
1100 (C-Cl).
N-[4-(2,2,6,6-Tetr am eth yl)piper idin yl]ph th alim ide (15).
Using 4-amino-2,2,6,6-tetramethylpiperidine (5 g, 33.76 mmol)
and phthalic anhydride (5 g, 32 mmol), compound 15 was
synthesized in the same way as 8 and was recrystallized from
methanol-water (1/1, v:v) (6.60 g, 72% yield): mp 276-278
°C; 1H NMR (CD3OD-d6) δ 1.63 (s, 6H, CH3), 1.67 (s, 6H, CH3),
2.07-3.35 (m, 5H, CH(CH2)2), 7.78-7.92 (m, 5H, aromatic);
13C NMR δ 24.72 (CH3), 30.59 (CH3), 30.59-58.87 (piperidine
C), 124.18-135.57 (aromatic C), 169.27 (CdO); IR (cm-1) 3520
(NH), 1780-1720 (imide), 1625 (phenyl).
58.81 (piperidine C), 105.34-144.16 (aromatic C); IR (cm-1
3440 (piperidine), 3350 (amino), 3300, 1650 (amide), 1620
(phenyl).
)
4-Nitr op h en yl-2,6-d im eth ylp h en ylth iou r ea (7). 2,6-
Dimethylaniline (3.04 g, 27.77 mmol) was solubilized in 3 mL
ethanol, and the mixture was heated under reflux. Phenyl
isothiocyanate (5 g, 27.75 mmol) dissolved in ethanol was
added dropwise over 30 min, and the reaction was pursued
for 1 h. The mixture was then poured onto ice, and the
resulting precipitate was filtered and purified by column
chromatography (silica gel, hexanes-ethyl acetate (3/1, v:v)
as elution solvent) yielding pure 7 (4.90 g, 60% yield): mp
1
178-180 °C; H NMR (DMSO-d6) δ 2.21 (s, 6H, CH3), 7.12-
8.23 (m, 7H, aromatic), 9.49 (s, 2H, NH); 13C NMR δ 18.12
(CH3), 120.93-146.55 (aromatic C), 180.24 (CdS); IR (cm-1
1600 (phenyl), 1500 (CdS), 1300 (nitro).
)
N-(2,6-Dim eth ylp h en yl)p h th a lim id e (8). A mixture of
2,6-dimethylaniline (3.43 g, 28.3 mmol) and phthalic anhydride
(5 g, 32 mmol) in acetic acid (20 mL) was stirred and heated
under reflux for 1 h. The solvent was evaporated in vacuo,
and the residual material was recrystallized from methanol-
water (1/1, v:v) yielding pure 8 (4.69 g, 66% yield): mp 206-
207 °C; 1H NMR (DMSO-d6) δ 2.08 (s, 6H, CH3), 7.20-8.13
(m, 6H, aromatic); 13C NMR δ 17.44 (CH3), 30.64 (CH3),
123.72-136.58 (aromatic C), 166.27 (CdO); IR (cm-1) 1780-
1720 (imide), 1600 (phenyl).
In Vivo Exp er im en ts. An im a ls. Male albino mice (CF-1
strain, 18-25 g; Charles River, Wilmington, MA) and male
albino rats (Sprague-Dawley, 100-150 g, Simonsen, Gilroy,
CA) were used as experimental animals. The animals were
4-Nitr o-N-(2,6-d im eth ylp h en yl)p h th a lim id e (9). Start-
ing from 2,6-dimethylaniline (3.43 g, 28.3 mmol) and 4-nitro-

3312 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Vamecq et al.
allowed free access to food (S/L Custom Lab Diet-7) and water,
except when removed from their cages for the experimental
procedures.
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Ack n ow led gm en t. The present work was supported
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(Faculty of Medicine and University Hospital of Lille,
France). J .V. is sponsored by the French INSERM. We
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