Synthesis and anticonvulsant properties of triazolo- and imidazopyridazinyl carboxamides and carboxylic acids

Article abstract of DOI:10.1016/S0968-0896(98)00057-1

Analogues of 3-amino-7-(2,6-dichlorobenzyl)-6-methyltriazolo[4,3-b]pyridazine PC25 containing amide or carboxylic acid function were synthesized and tested for anticonvulsant activity. The compounds having the imidazole ring substituted with an amide group have been found to be generally more active against maximal electroshock-induced seizures in mice (15.2≤ ED₅₀ ≤ 37.5mgkg^-1 orally). Furthermore, maximum activity was generally associated with a 2,6-dichlorobenzyl substitution pattern. 3-Amido-7-(2,6-dichlorobenzyl)-6-methyltriazolo[4,3-b]pyridazine 4b was also protective in the pentylenetetrazole-induced seizures test (ED₅₀=91.1mgkg^-1 orally) and blocked strychnine-induced tonic extensor seizures (ED₅₀=62.9mgkg^-1 orally). Moreover, calculated electrostatic isopotential maps of the whole active compounds were quite similar and, consequently, could be associated to optimum anticonvulsant activity. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00057-1

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 983±991
Synthesis and Anticonvulsant Properties of Triazolo- and
Imidazopyridazinyl Carboxamides and Carboxylic Acids
Stephane Moreau,a Pascal Coudert,a Catherine Rubat,b Danielle Vallee-Goyet,c
Daniel Gardette,c Jean-Claude Gramainc and Jacques Couqueleta,*
a
  Â
Groupe de Recherche en Pharmacochimie, Laboratoire de Chimie Therapeutique, Faculte de Pharmacie, Universite d'Auvergne,
28 Place Henri Dunant, 63 001 Clermont-Ferrand Cedex, France
b
Â
Laboratoire de Pharmacologie, Faculte de Pharmacie, Universite d'Auvergne, 28 Place Henri Dunant, 63 001 Clermont-Ferrand
Â
Cedex, France
 Á
Laboratoire de Chimie des Substances Naturelles, Universite Blaise Pascal CNRS UMR 6504, 63 177 Aubiere Cedex, France
c
Received 12 May 1997; accepted 10 February 1998
AbstractÐAnalogues of 3-amino-7-(2,6-dichlorobenzyl)-6-methyltriazolo[4,3-b]pyridazine PC25 containing amide or
carboxylic acid function were synthesized and tested for anticonvulsant activity. The compounds having the imidazole
ring substituted with an amide group have been found to be generally more active against maximal electroshock-
induced seizures in mice (15.2ꢀ ED50 ꢀ 37.5 mg kg 1 orally). Furthermore, maximum activity was generally associated
with a 2,6-dichlorobenzyl substitution pattern. 3-Amido-7-(2,6-dichlorobenzyl)-6-methyltriazolo[4,3-b]pyridazine 4b
was also protective in the pentylenetetrazole-induced seizures test (ED50=91.1 mg kg 1 orally) and blocked strychnine-
1
induced tonic extensor seizures (ED50=62.9 mg kg orally). Moreover, calculated electrostatic isopotential maps of
the whole active compounds were quite similar and, consequently, could be associated to optimum anticonvulsant
activity. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
activity of these new compounds. Moreover, structure±
activity relationships have been examined for these
molecules.
Previous works realized on 3-amino-7-(2,6-dichloro-
benzyl)-6-methyltriazolo[4,3-b]pyridazine PC25 and
analogues have shown some of these compounds to
be anticonvulsant agents with potent activity against
maximal electroshock-induced seizures (MES) in
mice.1 In order to increase this anticonvulsant activity,
®rstly an amide group or a carboxylic acid moiety was
introduced on the triazole or on the isosteric imidazole
ring of these molecules. Such substituents were chosen
owing to their presence in many anticonvulsants2±6
and specially in currently antiepileptic drugs namely
carbamazepine and valproic acid. Secondly, taking into
account our previous results,1 benzyl moiety attached to
the pyridazine ring was either unsubstituted or sub-
stituted by two chlorine atoms at the 2,6-position. We
report herein the synthesis and the anticonvulsant
Chemistry
Triazolo[4,3-b]pyridazines and imidazo[1,2-b]pyrid-
azines were synthesized according to Scheme 1. Starting
material 1 and key intermediates 2 and 5 were prepared
using reported methods.1,7 Hydrazinopyridazines 2 were
treated with ethyloxalyl chloride and the resulting
bicyclic esters 3 were hydrolysed in aqueous ammonia to
*Corresponding author. Tel.: 33 4 7360 8016; Fax: 33 4 7328
2707.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00057-1

984
S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991
Scheme 1.

S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991
985
lead to the amides 4. Synthesis of esters 6 and 9 was
achieved by reacting 3-aminopyridazines 5 with ethyl
2-chloroacetoacetate and ethyl bromopyruvate respec-
tively. Treatment of 6 and 9 with concentrated aqueous
ammonia furnished amides 7 and 10. Base-catalyzed
hydrolysis of esters 6 and 9, followed by acidi®cation to
pH 2 with 6 N hydrochloric acid, a€orded acids 8 and
11, respectively. Physical constants and spectral data of
compounds 4, 7, 8, 10 and 11 were reported in Tables 1
and 2.
serves as an excellent bioisostere of the triazolo[4,3-b]
pyridazine system with these anticonvulsant agents. On
the other hand, spontaneous motor activity was mea-
sured as a parameter of the sedative action of com-
pounds in the central nervous system (Table 3). Only
compound 10 was signi®cantly active, inducing a 38%
decrease in motor activity. In the other cases, either a
very slight increase or a decrease were observed in
spontaneous motor activity at 100 mg kg 1, contrary to
many anticonvulsant drugs (i.e., phenytoin, pheno-
barbital, diazepam, lamotrigine) that a€ect motor
movements at therapeutic doses.
Results and Discussion
Contained in Table 3 are the median doses for neuro-
logical impairment (TD50) using the rotorod test. Com-
pared with antiepileptic reference drugs, most pyridazine
derivatives were free of important neurotoxicity. Except
compounds 7b and 8a, the protection indices of test
drugs were largely greater than that of currently used
reference drugs. Considering their activity on the one
hand and their neurotoxicity on the other, the most
promising compounds were 4b, 7a, and 10 with PI
values of 20, over 52 and 17, respectively. Thus, these
three pyridazines were selected for determining their
ability to protect animals against chemically-induced
seizures. Among the possible mechanisms known to
inhibit seizure activity are the enhancement of inhibitory
(principally GABA-mediated) processes and the reduc-
tion of excitatory (particularly glutamate-mediated)
transmission.8,9 Involvement of one or two of these
mechanisms in anticonvulsant properties of the three
selected drugs was explored through classical empirical
animal models. Anticonvulsants which block seizures
induced by strychnine, bicuculline and N-methyl-d-l-
aspartate (NMDLA) do so by acting on glycine, g-amino-
butyric acid (GABA) and NMDA receptors, respec-
tively.10 With regard to anticonvulsants e€ective in the
yohimbine test, they may act at one and the same time
on GABA and NMDA receptors.10
The anticonvulsant activities of pyridazine derivatives 4,
7, 8, 10 and 11 were ®rst determined using the maximal
electroshock seizure (MES) test. The results were com-
pared to those obtained for PC251 and the clinically
proven antiepileptic agents: phenytoin, phenobarbital,
sodium valproate, carbamazepine, diazepam and lamo-
trigine. All compounds were orally administered to mice
and produced signi®cant anticonvulsant activity with
1
ED50's that ranged from 12.5 to 101 mg kg (Table 3).
Compared with sodium valproate whose ED50 value is
112 mg kg 1, pyridazine derivatives were more potent.
However, they were less active than PC25 and other
reference drugs in this screen. In this new series of
compounds, presence of an amide moiety compared
with a carboxylic acid function (e.g. 7a or 7b vs 8a or 8b
and 10 vs 11) resulted in an equipotent or an increase of
anticonvulsant activity. Furthermore, our previous
works1 have proved that the potencies of compounds
greatly increased if the benzyl moiety was substituted in
the ortho position with a chlorine atom. In the series of
amides and acids derivatives, the presence of such sub-
stituents also has, in two of the three cases, a signi®cant
impact on activity. Pyridazines 4a and 8a are respec-
tively at least ®ve- and threefold less active than the
2,6-dichloro analogues 4b and 8b. In addition, changing
the triazolopyridazine structure of PC25 or 4 into the
imidazo[1,2-b]pyridazine ring system present in 7, 8, 10
and 11, resulted in compounds having good activity
against MES. Thus, the imidazo[1,2-b]pyridazine bicycle
In the pentylenetetrazole (PTZ) test, hindlimb extension
was abolished by all pyridazine derivatives (Table 4). In
this test situation, ED50 values indicated that 4b, 7a, and
10 were less potent than PC25 and reference drugs such
as phenobarbital, carbamazepine, diazepam and lamo-
trigine. But they were more e€ective than the clinically
used drugs phenytoin and sodium valproate. The sc
PTZ test is fairly nonspeci®c and could clearly lead to
considerable ambiguity on the way of a drug exercising
its anticonvulsant e€ect. It can nevertheless be inferred
that test drugs may be active in treating generalized
absence seizures.11
Table 1. Physical constants of compounds 4, 7, 8, 10 and 11
Compd
R
mp Yield
(ꢁC) (%)
Formula
4a
4b
7a
7b
8a
8b
10
11
H
135
98
41
57
33
90
62
68
39
C
14H13N5O,H2O
2,6-diCl 243
230
2,6-diCl 250
260
C14H11N5C12O,2H2O
16H16N4O
16H14N4Cl2O
16H15N3O2
H
C
C
H
C
2,6-diCl 225
C16H13N3Cl2O2
C15H14N4O
Against strychnine-induced seizures, only 4b and 10 were
1
.
254
194
active with ED50 values equal to 61.2 and 93.1 mg kg
respectively, whereas phenytoin, phenobarbital and
C
15H11N3Cl2O2

986
S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991

S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991
987
Table 3. Locomotor activity, anticonvulsant activity (maximal electroshock seizure test), rotorod test and protective index for
pyridazine derivatives
PId
a,b,c
a,b
Compd
Locomotor activity at
1
MES ED50
1
Rotorod TD50
1
100 mg kg orallya
[( ) decrease, (+) increase]
(mg kg orally)
mg kg orally
4a
4b
+6‹3 (NS)
27‹5 (NS)
13‹7 (NS)
+4‹2 (NS)
9‹5 (NS)
13‹6 (NS)
38‹8e
69.7 (46.2±105.1)
12.5 (9.2±17.0)
15.2(8.3±27.8)
37.5(29.8±47.1)
101.0(67.4±151.4)
35.2(20.3±60.9)
19.7(12.3±31.3)
23.1(13.4±39.9)
9.2(6.3±13.1)
800.0 (560.2±1142.4)
253.3(171.2±374.8)
>800
11.5
20.3
>52.6
7.9
7a
7b
294.7(150.2±578.2)
881.5(726.7±1069.3)
483.3(331.7±704.2)
330.2(282.9±385.3)
294.7(150.2±578.2)
410.0(332.3±505.9)
30.5(22.7±40.9)
33.0(29.3±37.2)
208.0(173.8±248.9)
40.0(38.3±41.8)
2.8(1.8±4.2)
8a
8b
8.7
13.7
16.8
12.8
44.5
7.6
10
11
24‹6 (NS)
+16‹4 (NS)
16‹5e
PC25
Phenytoin
Phenobarbital
Sodium valproate
Carbamazepine
Diazepam
Lamotrigine
4.0(3.0±5.3)
4.6(3.0±7.0)
+33‹4e
7.2
1.9
5‹2 (NS)
+7‹3 (NS)
42‹6e
112.0(88.2±142.2)
5.5(4.6±6.5)
4.0(3.4±4.8)
7.3
0.7
34‹5e
3.9(2.2±7.1)
59.1(46.3±75.5)
15.1
aDrugs were administered 30 min before testing.
bMES (Maximal Electroshock Seizure Test).
cNumbers in parentheses are 95% con®dence intervals.
dPI (Protective Index)=TD50/ED50. PI values are for MES test.
eThe level of signi®cance was p<0.05. NS=not signi®cant.
1
Table 4. Anticonvulsant activity (ED50, mg kg orally) of pyridazine derivatives in the chemically induced seizures tests
Compd
Pentylenetetrazolea
Strychninea
Bicucullinea
Yohimbinea
NMDLAa
1
1
1
1
1
(85 mg kg sc)
(1 mg kg sc)
(0.8 mg kg iv)
(45 mg kg sc)
(345 mg kg sc)
4b
7a
91.1 (54.9±151.1)b
130.1(57.9±292.2)b
114.5(69.6±188.4)b
76.0(64.4±89.7)b
0c
15.0(13.4±16.8)b
20 (NS)c
17.5 (12.1±25.2)b
0.5 (0.3±0.8)b
42.1 (23.5±75.4)b
61.2(32.8±114.1)b
62.9(50.3±78.6)b
56.0(40.0±78.4)b
0c
93.0(55.2±156.7)b
0c
10(NS)c
0c
20(NS)c
0c
93.1(50.7±171.2)b
34.5(26.0±45.7)b
0c
40c,f
10
PC25
10(NS)c
0c
14(NS)d
0d
76.0(66.5±86.8)b
10(NS)d
17.0(12.1±23.9)b
10(NS)d
10(NS)d
2.3(1.4±3.7)b
10(NS)c
Phenytoin
Phenobarbital
Sodium valproate
Carbamazepine
Diazepam
Lamotrigine
0c
0c
20.0(14.7±27.2)b
14(NS)c
30.0(12.8±70.5)b
0d
8.5(7.6±9.6)b
1.1(0.6±1.8)b
13.0(4.7±35.8)b
0c
20(NS)d
30e,f
60c,f
0.4(0.2±0.6)b
21.3(16.3±27.8)b
aDrugs were administered 30 min before testing.
b95% con®dence intervals.
cPercentage of protection at 100 mg kg 1 orally.
dPercentage of protection at 150 mg kg 1 orally.
1
ePercentage of protection at 30 mg kg orally.
fThe level of signi®cance was p<0.05. NS=not signi®cant.
sodium valproate failed to protect mice from seizures.
But carbamazepine, diazepam and lamotrigine provided
better protection than the pyridazines did. In the bicu-
culline test, 4b and 7a were equipotent with ED50's of
62.9 and 56.0 mg kg 1, respectively, and were more
e€ective than PC25 (ED50=93.0 mg kg 1). Compound
10, as well as phenytoin, sodium valproate and carbam-
azepine were inactive in this animal seizure model.
Like phenytoin, sodium valproate, carbamazepine and
lamotrigine, 4b, 7a and 10 were unable to prevent sei-
zures induced by yohimbine when phenobarbital and
diazepam were very potent with ED50's values of 17.0
and 2.3 mg kg 1, respectively.
Finally, at the maximum doses employed, no protection
against seizures induced by NMDLA was noted, except

988
S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991
for marginal activity in the case of 7a. Only pheno-
barbital and lamotrigine exhibited prominent inhibition
of NMDLA-induced convulsions in mice.
imidazopyridazines 7a and 10 whose isopotential sur-
faces are quite di€erent because of their positive elec-
trostatic zone at the same position.
Therefore, except yohimbine and NMDLA induced
seizures, 4b was active against all of the seizure models
with a relatively narrow dose range and appeared to be
the most interesting anticonvulsant drug together with
compound PC25. Thus the anticonvulsant pro®le
observed for 4b was obviously somewhat better than
those observed for either phenytoin or sodium
valproate.
In conclusion, most of the compounds described in this
report have signi®cant activity as anticonvulsant drugs
in the MES screen, associated with high protection
As it has been demonstrated in a series of purine analo-
gues,12 the di€erences in activity of 4b, 7a, 10 and PC25
could be related to the electrostatic potential on the
surface of the molecules. In order to provide support for
this hypothesis, electrostatic isopotential maps of pyr-
idazines were generated using SYBYL 6.3 software13
from MOPAC charges (Figures 1±4) as described in
Experimental section. The major di€erences in the
shapes of the contour maps are observed near the 2-
position of the bicyclic pyridazine system. Optimum
activity could be associated with the electrostatic
potential surface presented by PC25. Triazolopyridazine
4b, which has a surface somewhat similar to that of
PC25, with a negative electrostatic isopotential near the
2-position of the pyridazine ring (see Scheme 1), is the
most active compound of this new series, contrary to
Figure 2. Stereoview of electrostatic isopotential surfaces (E) of
1
compound 7a. Eꢀ 5 kcal mol 1; bold solid. E ꢂ 5 kcal mol
;
solid.
Figure 1. Stereoview of electrostatic isopotential surfaces (E) of
1
Figure 3. Stereoview of electrostatic isopotential surfaces (E) of
1
compound 4b. Eꢀ 5 kcal mol 1; bold solid. E ꢂ 5 kcal mol
;
compound 10. E ꢀ 5 kcal mol 1; bold solid. E ꢂ 5 kcal mol
;
solid.
solid.

S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991
989
7 - Benzyl - 6 - methyltriazolo[4,3 - b]pyridazine - 3 -ethyl
carboxylate (3). To an anhydrous pyridine solution
(25 mL) of appropriate hydrazinopyridazine 2 (2.14 g,
10 mmol) was added ethoxalyl chloride (2.73 g,
20 mmol). The reaction mixture was stirred at room
temperature (2 h) and then re¯uxed (3 h). After cooling,
the solution was poured into water (100 mL) and
extracted with chloroform (3Â100 mL). The combined
extracts were dried (sodium sulfate) and evaporated in
vacuo. The residue was puri®ed by column chromato-
graphy on silica gel (ethyl acetate). Yield 18%, mp
94 ꢁC. IR (KBr) n cm 1: 1730,1600,1570. 1H NMR: 1.35
(t, 3H, 7.2 Hz, CH3), 2.45 (s, 3H, CH3), 4.15 (s, 2H,
CH2), 4.40 (q, 2H, 7.2 Hz, CH2), 7.25±7.40 (m, 5H,
C6H5), 8.05 (s, 1H, CH=).
7-Benzyl-6-methyl-triazolo[4,3-b]pyridazine-3-carbox-
amide (4a). To a suspension of 3 in 30% aqueous
ammonia (20 mL) was added ammonium chloride
(1.06 g, 20 mmol). The reaction mixture was stirred at
room temperature for 48 h. The precipitate formed was
®ltered o€, washed with water and recrystallized in an
ethanol :water mixture (75:25).
Figure 4. Stereoview of electrostatic isopotential surfaces
(E) of compound PC25. E ꢀ 5 kcal mol 1; bold solid. E ꢂ
5 kcal mol 1; solid.
For the synthesis of compounds 7 and 10, the same
procedure was carried out as described above.
indices. An amino and amido substituent in the 2-position
of the pyridazine derivatives ful®lls the structural
requirements for activity in the sc PTZ screen as well as
in the bicuculline and the strychnine tests. Furthermore,
electrostatic potential surfaces seem to be a useful tool
for selecting new synthetic targets, potentially active,
taking 4b or PC25 surfaces as a model.
7-Benzyl-2,6-dimethyl-imidazo[1,2-b]pyridazine-3-ethyl
carboxylate (6a). To a suspension of the appropriate
aminopyridazine 5 (2.68 g, 10 mmol) in anhydrous n-
butanol (35 mL) was added ethyl-2-choroacetoacetate
(4.94 g, 30 mmol). The reaction mixture was heated at
re¯ux for 30 h. After cooling, the aminopyridazine
hydrochloride precipitate formed was eliminated, the
®ltrate was evaporated in vacuo and the resulting resi-
due was puri®ed by column chromatography on silica
gel (ethyl acetate:hexane, 70:30). Yield 10%, mp 98 ꢁC.
IR (KBr) n cm 1: 3380, 1680, 1600, 1530. 1H NMR:1.35
(t, 3H, 7.2 Hz, CH3), 2.45 (s, 3H, CH3), 2.60 (s, 3H,
CH3), 4.10 (s, 2H, CH2), 4.30 (q, 2H, 7.2 Hz, CH2),
7.20±7.35 (m, 5H, C6H5), 7.75 (s, 1H, CH=).
Experimental
Chemistry
Melting points were taken on a Reichert apparatus and
were uncorrected. Infrared (M) spectra were run as
potassium bromide disks on a Beckman 4240 spectro-
photometer. The proton nuclear magnetic resonance
spectra were performed on a Bruker AC 400 (400 MHz).
Chemical shifts were reported in ppm related to internal
standard, tetramethylsilane. TLC was carried out on
Merck silica gel 60 F254 plates. Structures were
con®rmed by spectral data. Elemental analyses were
performed by the Service Central d'Analyses, Centre
National de la Recherche Scienti®que, 69 390 Ver-
naison, France, and all analytical results were within
‹0.4% of the theoretical values.
7-Benzyl-2,6-dimethyl-imidazo[1,2-b]pyridazine-3-carb-
oxylic acid (8a). To an aqueous ethanol (3 mL water +
10 mL ethanol) suspension of ester 6a (0.45 g,
1.46 mmol) was added a 10% aqueous solution of
sodium hydroxyde (5 mL). The reaction mixture was
stirred at room temperature (2 h) and then heated to
re¯ux (210 min). After cooling, the solution was acid-
i®ed to pH 2 with 6 N hydrochloric acid. The precipitate
formed was ®ltered o€, washed with water and recrys-
tallized in an ethanol :water mixture (50:50).
7-Benzyl-6-methyl-imidazo[1,2-b]pyridazine-2-ethyl
carboxylate (9a). To a suspension of the appropriate
Representative methods used to prepare pyridazine
derivatives are described for following examples.
aminopyridazine
5 (1.99 g, 10 mmol) in anhydrous

990
S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991
1
ethanol (12 mL) was added a solution of ethyl bromo-
pyruvate (1.95 g, 10 mmol) in anhydrous ethanol (12 mL).
The reaction mixture was stirred at room temperature for
1 h and then re¯uxed for 30 h. After ®ltration, the ®ltrate
was evaporated in vacuo and the resulting residue was
puri®ed by column chromatography on silica gel (ethyl
acetate). Yield 15%, mp 120 ꢁC. IR (KBr) n cm 1: 3400,
component induced by 85 mg kg
sc of pentylene-
tetrazole. Anticonvulsant activity was judged when the
component was blocked.
E€ect on seizures induced by strychnine.18 The ability of
the test compounds to provide a protection against
seizures was measured 30 min after administration. Pro-
tection was de®ned as the abolition of the hind leg tonic
extensor component of the seizure induced by 1 mg kg
sc injection of strychnine.
1
2940, 1730, 1660, 1600, 1490, 1450. H NMR: 1.35 (t,
1
3H, 7.2 Hz, CH3), 2.45 (s, 3H, CH3), 4.15 (s, 2H, CH2),
4.35 (q, 2H, 7.2 Hz, CH2), 7.25±7.40 (m, 5H, C6H5), 7.80
(s, 1H, CH=), 8.70 (s, 1H, CH=).
E€ect on seizures induced by bicuculline.19 The antagon-
ism of bicuculline-induced seizures was evaluated
according to a procedure similar to that described by
Heyer. Compounds were administered 30 min before the
test. Bicuculline was intravenously administered at a
dose of 0.8 mg kg 1. Protection against clonic seizures
was recorded.
The procedure used for the synthesis of acid 11, was the
same as described for compounds 8a.
Pharmacology
In the studies described below, all compounds were
orally administered in a 0.5% hydroxypropyl methyl
cellulose suspension to Swiss male mice purchased from
Depre (Saint-Doulchard, France) weighing 18±22 g.
Mice were kept in groups of ten in a temperature con-
trolled room with 12 h light/dark cycle. Twenty animals
were used in each test. Food and water were available
ad libitum until the time of experiment. The allocation
of animals in di€erent groups was randomized and
experiments were carried out in blind conditions.
E€ect on seizures induced by yohimbine. 20 For the
antagonism of yohimbine seizure studies, compounds
were administered 30 min before the test. Yohimbine
was subcutaneously administered at
a
dose of
45 mg kg 1. Animals that did not exhibit at least one
clonic seizure within 60 min were considered protected.
E€ect on seizures induced by NMDLA.21 Test com-
pounds were given 30 min prior to injection of
1
Sedative activity. This activity was evaluated by a
determination of spontaneous motor activity. The test
was performed by the method of Boissier and Simon14
in photoelectric activity cages (Apelex, Massy, France).
Test drugs were administered 30 min before evaluation
of spontaneous motor activity, and the number of pas-
sages was scored during 10 min.
345 mg kg s.c. of N-methyl-d,l-aspartate. Protection
against full generalized tonic seizures was recorded.
Data Analysis. Statistical analysis of the results was
performed using the method of Schwartz.22 The data on
the spontaneous motor activity were analyzed by using
the Student's t-test. All values were expressed as mean
‹SD. The data of electrically and chemically induced
seizures as well as the data of rotorod test were analyzed
by means of chi-square test with Yates correction. The
ED50 and TD50 values were determined using the
method of Litch®eld and Wilcoxon.23
Neurotoxicity. The rotorod test15 was used to evaluate
central nervous system toxicity. Test drugs were admi-
nistered 30 min before assay. Neurologic toxicity was
de®ned as the failure of the dosed animal to remain on a
3 cm diameter wood rod rotating at 6 rpm for 3 min.
Molecular modeling studies
Anticonvulsant activity. Compounds were tested for
their ability to protect mice against electrically and
chemically induced seizures.
The topographical and electrostatic characterization of
the studied molecules was performed using the SYBYL
6.3 software package on a Silicon Graphics Personal
IRIS 4D 35TG workstation. Structures were built
within SYBYL and minimized by MAXIMIN 2 Tripos
force ®eld, in vacuo conditions, to provide reasonable
standard geometries. Molecules were deemed to be
minimized when there was a minimum energy change of
less than 0.021 kJ mol 1 for one iteration. The conjugate
gradient method was used for minimization. All AM1
calculations involved the singlet state. Molecules were
deemed to be minimized when the gradient fell to less
than 0.021 kJ mol 1. The conformational space of PC25
E€ect on maximal electroshock seizures.16 Test drugs
and vehicles were orally administered 30 min before
subjecting the animals to maximal electroshock through
corneal electrodes. Protection against seizures was
de®ned as the abolition of the hind limb tonic extensor
component of seizures.
E€ect on seizures induced by pentylenetetrazole.16,17 Test
compounds were orally administered 30 min before
evaluation for their ability to prevent the tonic extensor

S. Moreau et al./Bioorg. Med. Chem. 6 (1998) 983±991
991
was explored as previously described.1 Compounds 4b,
7a and 10 were built by modi®cations of PC25 and
lowest energy conformers were obtained after rotation
of the amide or the acid group. These conformers were
submitted to AM1 calculations (MOPAC version 5.0)24
to optimize their geometry and determine atomic char-
ges distribution. The molecular electrostatic potential
surfaces were calculated in SYBYL and partitioned into
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Louis, MO 63144.
1
two intervals as follows: 5 kcal mol and down, and
1
+5 kcal mol and up.
14. Boissier J. R.; Simon, P. Arch. Int. Pharmacodyn. Ther.
1965, 158, 212.
15. Kinnard, W. J.; Carr, C. J. J. Pharmacol. Exp. Ther. 1957,
121, 354.
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22. Schwartz, D. In Methodes Statistiques
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a
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