3,5-Dihydro-4H-2,3-benzodiazepine-4-thiones: A new class of AMPA receptor antagonists

Article abstract of DOI:10.1021/jm9800393

Synthesis and evaluation of anticonvulsant activity of a series of 2,3- benzodiazepin-4-ones (2) chemically related to 1-(4'-aminophenyl)-4-methyl- 7,8-(methylenedioxy)-5H-2,3-benzodiazepine (1, GYKI 52466) have been reported in our recent publications. C

Full text of DOI:10.1021/jm9800393

J . Med. Chem. 1998, 41, 3409-3416
3409
3,5-Dih yd r o-4H-2,3-ben zod ia zep in e-4-th ion es: A New Cla ss of AMP A Recep tor
An ta gon ists
Alba Chimirri,*,† Giovambattista De Sarro,‡ Angela De Sarro,§ Rosaria Gitto,† Silvana Quartarone,†
Maria Zappala`,† Andrew Constanti,| and Vincenzo Libri|,
Dipartimento Farmaco-Chimico, Universita` di Messina, Viale Annunziata, 98168 Messina, Dipartimento di Medicina
Sperimentale e Clinica, Universita` di Catanzaro, Istituto di Farmacologia, Facolta` di Medicina, Universita` di Messina,
Dipartimento di Biologia, Universita` di Roma “Tor Vergata”, Italy, and Department of Pharmacology, School of Pharmacy,
University of London, WC1N 1AX, U.K.
Received J anuary 21, 1998
Synthesis and evaluation of anticonvulsant activity of a series of 2,3-benzodiazepin-4-ones (2)
chemically related to 1-(4′-aminophenyl)-4-methyl-7,8-(methylenedioxy)-5H-2,3-benzodiazepine
(1, GYKI 52466) have been reported in our recent publications. Compounds 2 manifested
marked anticonvulsant properties acting as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)-
propionic acid (AMPA) receptor antagonists. In an attempt to better define the structure-
activity relationships (SAR) and to obtain more potent and selective anticonvulsant agents,
1-aryl-3,5-dihydro-4H-2,3-benzodiazepine-4-thiones 3 were synthesized from the corresponding
isosteres 2. The evaluation is reported of their anticonvulsant effects, both in the audiogenic
seizures test with DBA/2 mice and against the maximal electroshock- and pentylenetetrazole-
induced seizures in Swiss mice. New derivatives 3 showed higher potency, less toxicity and
longer-lasting anticonvulsant action than those of the parent compounds 2 in all tests employed.
Analogous to derivatives 2, new compounds 3 do not affect the benzodiazepine receptor (BZR)
while they do antagonize AMPA-induced seizures; their anticonvulsant activity is reversed by
pretreatment with aniracetam but not with flumazenil, thus suggesting a clear involvement
of AMPA receptors. Electrophysiological data indicate a noncompetitive blocking mechanism
at the AMPA receptor sites for 3i, the most active of the series and over 5-fold more potent
than 1.
In tr od u ction
enedioxy-5H-2,3-benzodiazepine (1, GYKI 52466) act as
Glutamate, the neurotransmitter at most of the
excitatory synapses in the brain, activates a variety of
receptor subtypes that can be broadly divided into
ionotropic and metabotropic receptors. Ionotropic re-
ceptors mediate fast excitatory synaptic transmission
and, on the basis of pharmacological and molecular
biological studies, are named N-methyl-D-aspartate
(NMDA) and non-NMDA receptors.3 The non-NMDA
receptor group is further divided into 2-amino-3-(3-
hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA)
and kainate subtypes.
There is strong evidence that both NMDA and non-
NMDA receptors play an important role in acute neu-
rodegeneration and in convulsant phenomena.4 Hence,
all subtypes of these receptors are potential drug targets
for therapeutic intervention in a number of central
nervous system (CNS) diseases.5
highly selective noncompetitive antagonists at AMPA
receptors.10-13 They possess anticonvulsant properties
in various seizure models but, contrary to the classical
1,4-benzodiazepines, lack sedative-hypnotic activity
and do not bind to benzodiazepine receptors (BZR).14
In light of these pharmacological properties and as a
part of a program on potential anticonvulsant agents,15-17
our research group has been involved in designing new
compounds structurally related to 1.1 After the first
promising results and in an attempt to better define the
structure-activity relationships (SAR), we designed and
synthesized a wider series of 1-aryl-3,5-dihydro-4H-2,3-
benzodiazepin-4-ones 2 bearing suitably selected sub-
stituents on the heptatomic ring.2 By modification of
the substitution pattern, we obtained new potent anti-
convulsant agents which showed longer-lasting activity
and less toxicity than those of compound 1; moreover,
they proved to be selective AMPA receptor antagonists.2
Several previous studies have indicated that the
systemic administration of AMPA receptor antagonists
may be useful in the treatment of epilepsy and cerebral
ischemia6-9 and that some 2,3-benzodiazepine deriva-
tives such as 1-(4′-aminophenyl)-4-methyl-7,8-methyl-
* To whom correspondence should be addressed. Phone: +39 90
67676412. Fax +39 90 355613. E-mail: chimirri@pharma.unime.it.
† Dipartimento Farmaco-Chimico.
‡ Dipartimento di Medicina Sperimentale e Clinica.
§ Istituto di Farmacologia.
| Department of Pharmacology.
Dipartimento di Biologia.
S0022-2623(98)00039-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/08/1998

3410 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Chimirri et al.
Sch em e 1
With the goal of developing more potent and selective
compounds and improving the definition of the struc-
ture-activity relationships, we now report the synthesis
and biological activity of 1-aryl-3,5-dihydro-4H-2,3-
benzodiazepine-4-thiones 3a -k , a new class of benzo-
diazepine derivatives obtained from the corresponding
isosteres 2a -k , with the aim to explore the effects of
the lipophilicity on the anticonvulsant activity.
All synthesized compounds were tested against sound-
induced seizures in DBA/2 mice; this has been consid-
ered an excellent animal model for generalized epilepsy
and for screening new anticonvulsant drugs.18
Compounds 3a and 3i, the most active of the series,
were also evaluated both against pentylenetetrazole-
and maximal electroshock-induced seizures in Swiss
mice.
In addition, with the aim to confirm that their
anticonvulsant activity is mediated by the affinity for
AMPA receptor, we also evaluated 3a and 3i against
AMPA-induced seizures with or without a concomitant
treatment with aniracetam. Electrophysiological ex-
periments were conducted in order to investigate the
mode of inhibitory action at AMPA receptor sites, and
furthermore, the time course of anticonvulsant activity
was studied.
BZR affinity was assessed by the potencies of the 4H-
2,3-benzodiazepine-4-thiones to inhibit [3H]flumazenil
binding in a cortical membrane preparation, or by the
ability of flumazenil, a “neutral” BZR antagonist, to
reverse their anticonvulsant activity.
Ta ble 1. Anticonvulsant Activity of Compounds 1-3 against
Audiogenic Seizures in DBA/2 Mice and Relative Lipophilicity
(Rm)
ED50, µmol/kga ((95% confidence limits)
compd
clonic phase
tonic phase
Rm
The results obtained were compared to the findings
previously reported2 for compounds 2, and the resulting
modulation of anticonvulsant activity is described.
2a b
3a
33.9(26.0-44.2)
19.7(13.1-29.7)
102(76.1-137)
82.4(35.2-193)
110(79.3-151)
52.1(23.3-118)
>120
93.4(64.7-135)
>120
ND
37.8(23.7-60.1)
31.8(24.8-40.6)
15.0(8.60-26.1)
75.3(60.7-93.2)
55.0(36.0-84.2)
82.5(58.6-116)
33.3(15.2-73.1)
-0.244
0.014
0.012
0.244
0.035
0.269
-0.113
0.122
-0.135
0.092
-0.003
0.282
0.263
0.575
0.110
0.333
-0.630
-0.308
-0.542
-0.259
-0.421
-0.072
-0.308
2bb
3b
2cc
3c
Ch em istr y
Compounds 2a -k were prepared by a multistep
synthetic pathway according to a method previously
described.2 The target 1-aryl-3,5-dihydro-4H-2,3-ben-
zodiazepine-4-thiones 3a -k were obtained in good
yields by treatment with Lawesson’s reagent of com-
pounds 2a -k (Scheme 1).
The structures of all synthesized compounds were
determined by analytical and spectroscopic data (1H
NMR) and are reported in the Experimental Section.
2d c
3d
2ec
3e
>120
69.8(51.4-94.8)
>120
ND
26.7(14.7-48.2)
2fc
3f
30.6(19.6-44.7)
63.0(33.0-121)
105(52.0-194)
24.7(14.3-42.6)
38.0(20.0-72.0)
60.4(23.6-154)
2gc
3g
2h
3h
2ic
3i
>120
>120
103(73.6-144)
15.0(9.01-24.0)
6.30(2.60-15.4)
19.3(16.9-22.0)
18.8(8.70-36.5)
50.2(34.6-73.0)
29.8(21.4-41.4)
35.8(24.4-52.4)
81.5(63.3-105)
12.6(8.01-19.0)
3.30(1.30-8.30)
18.3(16.0-20.8)
9.10(3.70-22.3)
43.7(31.3-61.0)
20.3(13.6-30.5)
25.3(16.0-40.0)
2jc
3j
Resu lts a n d Discu ssion
2k c
3k
1
1-Aryl-3,5-dihydro-4H-2,3-benzodiazepine-4-thiones
3a -k were evaluated after intraperitoneal (ip) admin-
istration in DBA/2 mice, a strain genetically susceptible
to sound-induced seizures,18 and the results were com-
pared with the anticonvulsant properties of the corre-
sponding 4H-2,3-benzodiazepin-4-ones 2a -k and 1.
Table 1 reports the median effective dose (ED50) values
required to prevent clonic and tonic phases of seizures.
The rank order of anticonvulsant potency was as fol-
lows: 3i > 2i > 3j > 3a > 2j > 3k > 3f > 2a > 1 > 2f
> 2k > 3c > 2g > 3b > 3d > 2b > 3h > 3g > 2c. The
remaining compounds showed no activity.
The introduction of a thiocarbonyl group at the C-4
position of the heptatomic ring generally leads to new
compounds 3 with anticonvulsant properties higher
than those of the carbonyl isostere analogues 2. In
particular, 1-(4′-aminophenyl) derivative 3i is 2.5-fold
more active than the corresponding 2i.
a
All data were calculated according to the method of Litchfield
and Wilcoxon.32 At least 32 animals were used to calculate each
ED50 value. Reference 1. c Reference 2. ND ) not detectable.
b
Analogous to the 2 series, in the new thiocarbonyl
derivatives 3, the presence of a methyl group at N-3 led
to compounds (i.e., 3f, 3g, 3h , and 3k ) with anticonvul-
sant properties weaker than those of N-3 unsubstituted
derivatives.
The different anticonvulsant potencies of the 1-aryl-
3,5-dihydro-4H-2,3-benzodiazepine-4-thiones may be cor-
related to their relative lipophilicities (Table 1), which
positively influence the cellular permeability and, there-
fore, the penetration through the blood-brain barrier.
Unexpectedly, the 3′-nitrosubstituted derivative 3e
induced clonic jerks in DBA/2 mice. There is no

3,5-Dihydro-4H-2,3-benzodiazepine-4-thiones
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3411
Ta ble 2. Anticonvulsant Activity against the MES- and
PTZ-Induced Seizures in Swiss Mice of 2a , 3a , 2i, 3i, and 1
Ta ble 3. ED50 Values of 2a , 3a , 2i, 3i, and 1 against the
Clonic and Tonic Seizures Induced by icv Injection of AMPA in
DBA/2 Micea
ED50, µmol/kg ((95% confidence limits)a
ED50, µmol/kg ((95% confidence limits)
compd
MES
PTZ
compd
clonic phase
tonic phase
2a b
3a
2ic
3i
35.8(28.6-44.7)
27.8(21.5-35.9)
15.9(7.3-33.5)
7.75(3.89-15.4)
35.7(29.3-43.4)
68.2(54.6-85.2)
33.7(18.4-61.6)
22.6(11.7-43.8)
15.4(7.00-33.9)
68.3(56.2-83.1)
2a b
3a
2ic
3i
66.0(45.9-94.9)
43.1(29.1-63.8)
32.1(23.2-44.3)
17.1(7.70-38.0)
57.5(43.5-76.0)
42.6(26.4-68.8)
31.1(19.3-50.2)
25.0(16.5-30.0)
11.9(4.60-30.8)
40.5(26.3-60.8)
1
1
a
All data were calculated according to the method of Litchfield
and Wilcoxon.32 At least 32 animals were used to calculate each
a
AMPA was administered icv at the CD97 for either clonus (9.7
nmol) or forelimb tonic extension (11.7) 30 min after injection of
tested compounds. All data were calculated according to the
method of Litchfield and Wilcoxon.32 At least 32 animals were used
ED50 value. Reference 1. c Reference 2.
b
to calculate each ED50 value. Reference 1. c Reference 2.
b
Thiocarbonyl derivatives 3 could undergo biotransfor-
mation into the corresponding compounds 2 (as observed
by preliminary HPLC studies on rat plasma) acting at
first as unaltered and then as carbonyl isosters 2.
The hypothesis that the derivatives of compounds 3
interact with non-NMDA receptors was also confirmed
by suitable tests on some of them. Analogous to parent
compounds 2, 3a and 3i afforded effective protection
against seizures induced by intracerebroventricular (icv)
administration of AMPA, a chemical agent which stimu-
lates the AMPA/kainate receptor complex (Table 3). The
clonic and tonic phases of the AMPA-induced seizures
were significantly reduced 30 min after ip administra-
tion of 3a , 3i, and 1 as shown in Figure 2.
In the present study we also demonstrated that
aniracetam, a potentiator of AMPA effects,19 markedly
antagonized the anticonvulsant effects of 3a and 3i in
DBA/2 mice (Table 4) with a pattern of activity similar
to that of 1, considered as AMPA/kainate receptor
antagonist.10c Since it has been shown that aniracetam
is a positive allosteric modulator of AMPA/kainate-
selective glutamate receptors, we therefore suggest that,
by analogy to 1,10d,11 2a , and 2i,2 also the 4H-2,3-
benzodiazepine-4-thiones 3a and 3i described here
might antagonize the AMPA/kainate receptor-mediated
responses by an allosteric blocking mechanism.
In addition, the anticonvulsant properties of deriva-
tives 3a and 3i were not significantly affected by the
concomitant treatment with flumazenil (Table 5). The
results obtained in vivo were corroborated by in vitro
binding experiments in which the potency of the deriva-
tives 3 as inhibitors of [3H]flumazenil binding to mem-
branes from cortex was evaluated, and no inhibition was
observed (IC50 > 10 000 nM).
Moreover, binding experiments showed that com-
pounds 3 failed to displace [3H]spiperone from dopamine
and 5-hydroxytryptamine1 receptors, [3H]ketanserin
from 5-hydroxytryptamine2 receptors, [125I]pindolol from
â-adrenergic receptors, [3H](RS)-3-(2-carboxypiperazin-
4-yl)-propyl-1-phosphonic acid ([3H]CPP) and [3H]dizo-
cilpine ([3H]MK-801) from NMDA receptors, [3H]5,7-
dichlorokynurenic acid ([3H]5,7-DCKA) from the glycine
site on NMDA receptors, [3H]AMPA and [3H]6-cyano-
7-nitroquinoxaline-2,3-dione ([3H]CNQX) from AMPA/
kainate receptors, and a mixture of [3H](1S,3R)ACPD
and [3H](1R,3S)ACPD from metabotropic glutamate
receptors. Despite the lack of activity at the glycine site
on NMDA receptors and dopamine, serotonin, norad-
renaline, NMDA, and metabotropic glutamate binding
sites an interaction at other sites involved in the
F igu r e 1. Anticonvulsant effects of 2a , 3a , and 1 (33 µmol/
kg ip) plotted against audiogenic seizures in DBA/2 mice. The
ordinate shows seizure score, the abscissa shows the time after
intraperitoneal administration of drug in min. For the deter-
mination of each point 10 animals were used. The latter
represents the mean seizure score ((SEM) against audiogenic
seizures in DBA/2 mice.
explanation for this discrepancy, and one can only
speculate that this may be due to a change in the
interaction mode of 3e which probably might act as an
AMPA receptor agonist or bind to a different receptor
site.
The most active derivatives of the series, 3a and 3i,
their parent compounds, 2a and 2i, and 1 were further
evaluated against seizures induced by maximal elec-
troshock (MES) and the administration of pentylene-
tetrazole (PTZ), and the biological results are reported
below.
As shown in Table 2, the tonic extension of the
seizures induced by MES and the clonic phase of the
seizures induced by PTZ were significantly reduced 45
min after ip administration of 2a , 2i, 3a , 3i, and 1 with
the same rank order of anticonvulsant potency observed
in the test of audiogenic seizures.
Following ip administration of some active compounds
such as 2a and 3a , maximum protection was observed
from 45 to 90 min for 2a and from 45 to 120 min for
compound 3a with subsequent return to control seizure
response at 180 min for derivative 2a and at 240 min
for compound 3a , as shown in Figure 1. In contrast, 1
displayed maximum protection from 5 to 15 min fol-
lowed by gradual return to control seizure response
between 30 and 90 min (Figure 1).
The longer-lasting anticonvulsant activity of com-
pounds 3a could be explained taking into account also
the influence of biotransformation reactions on molec-
ular properties and the consequences of such changes.

3412 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Chimirri et al.
Ta ble 5. ED50 Values of 2a , 3a , 2i, 3i, and 1 against the
Clonic and Tonic Phases of the Audiogenic Seizures after
Concomitant Treatment with Flumazenil in DBA/2 Micea
ED50, µmol/kg (( 95% confidence limits)
Flumazenil,
compd
µmol
clonic phase
tonic phase
2a b
8.24
24.72
8.24
24.72
8.24
24.72
8.24
24.72
8.24
38.2(28.5-51.2)
50.9(36.9-70.1)
22.4(17.9-28.0)
19.3(16.9-22.0)
14.1(10.1-19.9)
12.0(6.82-21.0)
6.90(4.70-10.2)
7.10(5.60-9.00)
37.8(23.7-60.1)
39.5(29.6-53.7)
35.8(26.2-49.0)
41.3(28.0-60.7)
13.6(7.40-25.2)
12.6(8.01-19.0)
12.0(6.82-21.0)
10.2(7.61-13.7)
3.24(1.24-4.85)
32.2(2.71-4.57)
26.7(14.7-48.2)
29.7(20.9-42.1)
3a
2ic
3i
1
24.72
a
All data were calculated according to the method of Litchfield
and Wilcoxon.32 At least 32 animals were used to calculate each
b
ED50 value. Reference 1. c Reference 2.
dose-response relationship in a nonparallel manner,
even at the highest agonist concentration used (5 µM).
This noncompetitive mode of antagonism of AMPA
responses by 3i detected here (Figure 3), is in agreement
both with previous in vitro data obtained in our labora-
tory with compound 2i and 12 and with other data
observed from cultured hippocampal neurons,10d,11b
cortical wedges,12a and hippocampal slices.12b
As shown in Figure 3, in the presence of a fixed
concentration (50 µM) of 3i, the peak amplitude of
AMPA-induced inward currents was markedly reduced
at each agonist dose level (∼75-85% suppression) with
a clear noncompetitive-type depression of the AMPA
dose-response curve. These data confirm that deriva-
tives 2i, 3i, and 1 may all share a common noncompeti-
tive mode of action at the AMPA receptor.
Although it has long been known that antagonists of
the excitatory amino acids (EAA) acting on NMDA
receptors, especially those which block ion channels
[e.g., dizocilpine (MK-801)], can induce cognitive deficits
and a variety of other neurological and behavioral side
effects,11a,20 there are studies showing that potent
antagonists at the AMPA/kainate receptor have anti-
convulsant effects at doses below those impairing be-
havior.21
The anticonvulsant activity of the title compounds
was evident at doses which generally did not cause
sedation and ataxia. The therapeutic index (TI) of the
most active compounds 3a and 3i is 2.5-3 times that
of compound 1 (Table 6).
In conclusion, the anticonvulsant activity profile and
improved therapeutic index of some of the new 1-aryl-
3,5-dihydro-4H-2,3-benzodiazepine-4-thiones 3 provide
an interesting illustration of the research effort which
is being made to discover new drugs interacting with
AMPA receptors and possessing therapeutic potential
with lower side effects. Extensive further modifications
are in progress and will be used to challenge and refine
our pharmacophore model.
F igu r e 2. Anticonvulsant effects of 3a , 3i, and 1 plotted
against seizures induced by AMPA in DBA/2 mice. The
ordinate shows percent of response of clonic (A) or tonic (B)
seizures, the abscissa shows the dose in µmol/kg ip. For the
determination of each point, 10 animals were used.
Ta ble 4. ED50 Values of 2a , 3a , 2i, 3i, and 1 against the
Clonic and Tonic Phases of the Audiogenic Seizures after
Pretreatment with Aniracetam in DBA/2 Micea
ED50, µmol/kg ((95% confidence limits)
compd
clonic phase
tonic phase
2a b
3a
2ic
3i
215(142-324)*
98.0(64.4-149)*
65.4(44.5-96.2)*
46.1(34.6-61.3)*
134(88.8-203)*
157(114-217)*
69.1(42.4-113)*
58.2(43.4-77.9)*
38.5(26.9-55.0)*
100(63.4-158)*
1
a
All data were calculated according to the method of Litchfield
and Wilcoxon.32 At least 32 animals were used to calculate each
ED50 value. Significant differences between ED50 values of the
group treated with aniracetam + 2,3-benzodiazepine and the group
treated with 2,3-benzodiazepine alone (Table 1) are denoted *P <
b
0.01. Reference 1. c Reference 2.
Exp er im en ta l Section
generation or expression of seizures cannot presently
be ruled out.
Ch em istr y. Melting points were determined on a Kofler
hot stage apparatus and are uncorrected. Elemental analyses
(C, H, and N) were carried out on a Carlo Erba model 1106
elemental analyzer, and the results are within (0.4% of the
theoretical values. Merck silica gel 60 F254 plates were used
for analytical TLC; column chromatography was performed on
Merck silica gel 60 (70-230 mesh). 1H NMR spectra were
Indeed, the noncompetitive nature of the AMPA block
exerted by the most potent derivative tested, 3i, was
confirmed in electrophysiological experiments performed
in olfactory cortical brain slice neurons. In these tests,
3i, similar to 2i and 1, consistently depressed the AMPA

3,5-Dihydro-4H-2,3-benzodiazepine-4-thiones
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3413
Ta ble 6. ED50 and TD50 Values (95% Confidence Limits) of
Compounds 1-3 on Locomotion Assessed by Rotarod Test
Following 30 min Pretreatmenta
ED50, µmol/kg
clonic phase
TD50, µmol/kg
locomotor deficit
TId
TD50/ED50
compd
2a b
3a
33.9(26.0-44.2)
19.7(13.1-29.7)
102(76.1-137)
82.4(35.2-193)
110(79.3-151)
52.1(23.3-119)
142(87.3-231)
98.9(16.0-611)
240(102-564)
193(169-220)
226(116-437)
73.0(56.2-95.0)
>150
4.2
5.02
2.3
3.26
2.0
1.4
ND
ND
4.1
3.17
3.8
6.76
2.7
4.08
3.2
2bc
3b
2cc
3c
2d c
3d
2fc
3f
>120
93.4(64.7-135)
37.8(23.7-60.1)
30.6(19.6-44.7)
15.0(9.01-24.0)
6.30(2.60-15.4)
19.3(16.9-22.0)
18.8(8.10-36.5)
50.2(34.6-73.0)
29.8(21.4-41.4)
35.8(24.4-52.4)
>150
154(104-227)
97.1(73.2-129)
56.8(39.3-82.1)
42.6(26.4-68.8)
51.5(34.1-77.8)
76.7(63.3-93.0)
159(95.0-268)
130(85.4-197)
76.1(47.5-122)
2ic
3i
2jc
3j
2k c
3k
1
4.35
2.1
a
All data are expressed as µmol/kg and were calculated
according to the method of Litchfield and Wilcoxon.32 At least 32
animals were used to calculate each ED50 and TD50 value.
b
d
Reference 1. c Reference 2. TI ) Therapeutic Index and repre-
sents the ratio between TD50 and ED50 (from the clonic phase of
the audiogenic seizures); ND ) not determined.
(3a -k ). Lawesson’s Reagent [2,4-bis(4-methoxyphenyl)-2,4-
dithioxo-1,3,2,4-dithiadiphosphethane] (0.55 mmol) was added
to a solution in dry toluene (150 mL) of the suitable 2,3-
benzodiazepin-4-one derivative (2a -k , 1.00 mmol), which was
prepared according to a procedure previously described.2 The
mixture was heated at 120 °C for 90 min, and the course of
the reaction was monitored by TLC. The solution was then
cooled at room temperature and filtered. The solvent was
removed under reduced pressure, and the resulting oily yellow
residue was treated with EtOH to provide compound 3, which
was recrystallized from EtOH (derivatives 3a -h ) or purified
by column chromatography with diethyl ether/light petroleum
(80/20) as eluant (derivatives 3j-k ).
3,5-Dih yd r o-7,8-d im eth oxy-1-p h en yl-4H-2,3-ben zod ia z-
ep in e-4-th ion e (3a ): yellow crystals, mp 166-169 °C, yield
65%; 1H NMR 3.71 (s, 3H, OCH3-8), 3.99 (s, 3H, OCH3-7), 3.97
(bs, 2H, CH2), 6.35 (s, 1H, H-9), 6.88 (s, 1H, H-6), 7.42-7.65
(m, 5H, Ar), 10.02 (bs, 1H, NH). Anal. (C17H16N2O2S) C, H,
N.
1-(4′-Ch lor op h en yl)-3,5-d ih yd r o-7,8-d im eth oxy-4H-2,3-
ben zod ia zep in e-4-th ion e (3b): yellow crystals, mp 222-225
°C, yield 82%; 1H NMR 3.73 (s, 3H, OCH3-8), 3.99 (s, 3H,
OCH3-7), 3.91 (bs, 2H, CH2), 6.59 (s, 1H, H-9), 6.87 (s, 1H,
H-6), 7.40-7.60 (m, 4H, Ar), 10.13 (bs, 1H, NH). Anal.
(C17H15ClN2O2S) C, H, N.
F igu r e 3. Noncompetitive-type antagonism of AMPA-induced
inward currents by 2,3-benzodiazepine derivatives, recorded
in guinea pig olfactory cortical neurons voltage-clamped at -80
mV (in the presence of 1 µM TTX). (A) Pooled dose-response
curves to AMPA measured in the absence (O; N ) 4) and
presence (b; N ) 4) of 50 µM compound 3i (upper panel) or in
the absence (4; N ) 3) and presence (1; N ) 3) of 50 µM 1
(lower panel). Each point represents the mean ((SEM) plotted
against the AMPA concentration (0.25-5 µM, log scale). Points
without error bars had a SEM less than the symbol size. (B)
Chart records showing inward currents activated by a 4-min
bath application of AMPA (2 µM) in control and after a 15-
min preincubation with 50 µM compound 3i (upper panel) or
50 µM 1 (lower panel); note the similar degree of response
depression induced by both compounds. The depression of 1,
2, and 5 µM AMPA mean currents by either antagonist was
significant relative to control (P < 0.05, 0.01, and 0.001,
respectively; Student’s t test). Scale bar applies to all traces.
Drug washout periods were recorded at 0.4 times the chart
speed.
1-(4′-Br om op h en yl)-3,5-d ih yd r o-7,8-d im eth oxy-4H-2,3-
ben zod ia zep in e-4-th ion e (3c): yellow crystals, mp 115-118
°C, yield 79%; 1H NMR 3.73 (s, 3H, OCH3-8), 3.99 (s, 3H,
OCH3-7), 3.91 (bs, 2H, CH2), 6.59 (s, 1H, H-9), 6.87 (s, 1H,
H-6), 7.50-7.60 (m, 4H, Ar), 9.89 (bs, 1H, NH). Anal. (C17H15
BrN2O2S) C, H, N.
-
3,5-Dih yd r o-7,8-d im et h oxy-1-(4′-n it r op h en yl)-4H -2,3-
ben zod ia zep in e-4-th ion e (3d ): yellow crystals, mp 228-230
°C, yield 65%, 1H NMR 3.72 (s, 3H, OCH3-8), 4.00 (s, 3H,
OCH3-7), 3.97 (bs, 2H, CH2), 6.53 (s, 1H, H-9), 6.88 (s, 1H,
H-6), 7.83-8.31 (m, 4H, Ar), 10.20 (bs, 1H, NH). Anal.
(C17H15N3O4S) C, H, N.
3,5-Dih yd r o-7,8-d im et h oxy-1-(3′-n it r op h en yl)-4H -2,3-
ben zod ia zep in e-4-th ion e (3e): yellow crystals, mp 256-258
°C, Yield 57%; 1H NMR 3.72 (s, 3H, OCH3-8), 4.01 (s, 3H,
OCH3-7), 3.99 (bs, 2H, CH2), 6.57 (s, 1H, H-9), 6.90 (s, 1H,
H-6), 7.62-8.54 (m, 4H, Ar), 10.06 (bs, 1H, NH). Anal.
(C17H15N3O4S) C, H, N.
recorded in CDCl3 on a Varian-Gemini-300 spectrometer.
Chemical shifts were expressed in δ (ppm) relative to TMS as
internal standard and coupling constants (J ) in Hz. All
exchangeable protons were confirmed by addition of D2O.
Gen er a l P r oced u r e for t h e Syn t h esis of 1-Ar yl-3,5-
d ih yd r o-7,8-d im eth oxy-4H-2,3-ben zod ia zep in e-4-th ion es
3,5-Dih yd r o-7,8-d im eth oxy-3-m eth yl-1-p h en yl-4H-2,3-
ben zod ia zep in e-4-th ion e (3f): yellow crystals, mp 160-162
°C, Yield 75%; 1H NMR 3.73 (s, 3H, OCH3-8), 3.99 (s, 3H,

3414 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Chimirri et al.
OCH3-7), 3.73 and 4.18 (dd, 2H, J ) -12.6, CH2), 3.81 (s, 3H,
NCH3), 6.64 (s, 1H, H-9), 6.91 (s, 1H, H-6), 7.43-7.67 (m, 5H,
Ar). Anal. (C18H18N2O2S) C, H, N.
Ma xim a l Electr osh ock Seizu r e Test in Sw iss Mice.
Electrical stimuli were applied via ear-clip electrodes to Swiss
mice (rectangular constant current impulses, amplitude 50
mA, width 20 ms, frequency 35 Hz, duration 400 ms) according
to the method of Swinyard et al.25 Abolition of tonic hindlimb
extension after drug treatment was considered as the endpoint
of protection. In general, the dose-response curves were
estimated by testing four to five doses using 8-10 mice for
each dose.
1-(4′-Br om oph en yl)-3,5-dih ydr o-3-m eth yl-7,8-dim eth oxy-
4H-2,3-ben zod ia zep in e-4-th ion e (3g): yellow crystals, mp
1
168-170 °C, yield 66%; H NMR 3.75 (s, 3H, OCH3-8), 3.99
(s, 3H, OCH3-7), 3.71 and 4.19 (dd, 2H, J ) -12.6, CH2), 3.80
(s, 3H, NCH3), 6.61 (s, 1H, H-9), 6.90 (s, 1H, H-6), 7.52-7.62
(m, 4H, Ar). Anal. (C18H17BrN2O2) C, H, N.
3,5-Dih yd r o-7,8-d im eth oxy-3-m eth yl-(4′-n itr op h en yl)-
4H-2,3-ben zod ia zep in e-4-th ion e (3h ): yellow crystals, mp
228-230 °C, yield 45%; H NMR 3.73 (s, 3H, OCH3-8), 4.00
P en tylen etetr a zole-In d u ced Seizu r es in Sw iss Mice.
Male Swiss mice (20-26 g, 42-48 days old) were purchased
from Charles River (Calco, Como, Italy) and were pretreated
with vehicle or drug 45 min before the subcutaneous (sc)
administration of pentylenetetrazole. For systemic injections,
all tested compounds were given ip (0.1 mL/10 g of body weight
of the mouse) as a freshly prepared solution in 50% DMSO
and 50% sterile saline (0.9% NaCl). The convulsive dose 97
(CD97) of PTZ (85 mg/kg) was applied, and the animals were
observed for 30 min. A threshold convulsion was an episode
of clonic spasms lasting for at least 5 s. The absence of this
threshold convulsion over 30 min indicated that the tested
substance had the ability to elevate the PTZ seizure thres-
hold.26
1
(s, 3H, OCH3-7), 3.72 and 4.09 (dd, 2H, J ) -12.4, CH2), 3.84
(s, 3H, NCH3), 6.55 (s, 1H, H-9), 6.92 (s, 1H, H-6), 7.83-8.32
(m, 4H, Ar). Anal. (C18H17N3O4S) C, H, N.
1-(4′-Am in op h en yl)-3,5-d ih yd r o-7,8-d im eth oxy-4H-2,3-
ben zod ia zep in e-4-th ion e (3i): yellow powder, mp 225-227
°C, yield 50%; 1H NMR 3.75 (s, 3H, OCH3-8), 3.99 (s, 3H,
OCH3-7), 3.96 (bs, 4H, NH2 and CH2), 6.68 (s, 1H, H-9), 6.72
(s, 1H, H-6), 6.69-7.46 (m, 4H, Ar), 9.80 (bs, 1H, NH). Anal.
(C17H17N3O2S) C, H, N.
1-(3′-Am in op h en yl)-3,5-d ih yd r o-7,8-d im eth oxy-4H-2,3-
ben zod ia zep in e-4-th ion e (3j): yellow powder, mp 128-130
°C, yield 58%; 1H NMR 3.74 (s, 3H, OCH3-8), 3.99 (s, 3H,
OCH3-7), 3.90 (bs, 4H, NH2 and CH2), 6.68 (s, 1H, H-9), 6.86
(s, 1H, H-6), 6.80-7.24 (m, 4H, Ar), 9.87 (bs, 1H, NH). Anal.
(C17H17N3O2S) C, H, N.
AMP A-In d u ced Seizu r es in DBA/2 Mice. Seizures were
also induced by icv injection of AMPA. The CD50 of AMPA for
clonus was 1.76 (1.06-3.07), while that for tonus was 2.90
(1.83-4.58) nmol. For icv injection, mice were anesthetized
with diethyl ether, and injections were made in the left or right
lateral ventricle (coordinates 1 mm posterior and 1 mm lateral
to the bregma; depth 2.4 mm) using a 10 µL Hamilton
microsyringe (type 701N) fitted with a nylon cuff on the needle
as previously described:27 injections of drugs by this procedure
led to a uniform distribution throughout the ventricular system
within 10 min. The animals were placed singly in a 30 × 30
× 30 cm box, and the observation time was 30 min after the
administration of AMPA.
1-(4′-Am in op h en yl)-3,5-d ih yd r o-7,8-d im eth oxy-3-m eth -
yl-4H-2,3-ben zod ia zep in e-4-th ion e (3k ): yellow powder, mp
1
182-185 °C, yield 40%; H NMR 3.75 (s, 3H, OCH3-8), 3.98
(s, 3H, OCH3-7), 3.71 and 4.07 (dd, 2H, J ) -12.4, CH2), 3.76
(s, 3H, NCH3), 3.92 (bs, 2H, NH2), 6.68 (s, 1H, H-9), 6.89 (s,
1H, H-6), 6.70-7.47 (m, 4H, Ar). Anal. (C18H19N3O2S) C, H,
N.
Lip op h ilicity Mea su r em en ts. The relative lipophilicity
(Rm) of the compounds was measured by reversed-phase high-
performance thin-layer chromatography (RP-HPTLC) accord-
ing to the method previously described.16a,22 Briefly, Whatman
KC18F plates were used as the nonpolar stationary phase. The
plates were dried at 105 °C for 1 h before use. The polar
mobile phase was a 2:1 (v/v) mixture of acetone and water.
Each compound was dissolved in CHCl3 (3 mg/mL), and 1 µL
of solution was applied onto the plate. The experiments were
repeated five times with different disposition of the compounds
on the plate. The Rf values were expressed as the mean values
of the five determinations. The Rm values were calculated from
Mem br an e P r epar ation an d [3H]Flu m azen il an d Oth er
Bin d in g Stu d ies. Male SD/Rij rats (FRAR, S.Pietro al
Natisone, UD, Italy) weighing 200-250 g were decapitated,
and different brain areas were rapidly dissected on ice. Brain
regions were homogenized in 20 mL of ice-cold 0.32 M sucrose,
pH 7.4, by using a glass homogenizer with a Teflon pestle (10
up and down strokes). The homogenate was centrifuged at
1000g at 4 °C for 10 min, the P1 pellet was discarded, and the
supernatant was collected and recentrifuged at 20000g at 4
°C for 20 min. The resulting crude mitochondrial pellet (P2)
was resuspended in 20 mL of ice-cold distilled water and
homogenized. The homogenate was centrifuged at 8000g at
4 °C for 20 min, the supernatant was collected and recentri-
fuged at 48000g at 4 °C for 20 min, and the final crude
microsomal pellet (P3) was frozen for at least 24 h. The pellet
was resuspended in 10 mL of 50 mM Tris-HCl pH 7.4,
centrifuged at 48000g at 4 °C for 20 min, and then resuspended
in 10 vol of the same buffer for the standard binding assay.
Aliquots of membrane suspensions (100 µL or 0.15 mg of
protein) were added to the incubation medium containing 1
nmol of [3H]flumazenil (specific activity 72.4 Ci/mmol) in a
final volume of 1 mL of 50 mM Tris-HCl, 120 mM NaCl, and
5 mM KCl, pH 7.4. All compounds were dissolved in DMSO
at a final concentration of 1%. Incubations were carried out
for 60 min at 4 °C in triplicate, and nonspecific binding was
measured in the presence of 10 µM diazepam. Reactions were
stopped by the addition of 5 mL of ice-cold Tris-HCl followed
by rapid filtration through Whatman GF/C glass fiber filters
(Whatman Inc. Clifton, NJ ) and two additional washes. The
radioactivity trapped on the filters was counted after the
addition of 8 mL of Filter Count (Packard) by liquid scintil-
lation spectrometry. The experiments were run in triplicate
with eight different concentrations of competing ligand. The
possibility that compounds 3 bind to receptors other than BZR
was studied in crude rat brain synaptic membranes according
to established protocols28 by using [3H]spiperone, [3H]ket-
anserin, [125I]pindolol, [3H]CPP, [3H]MK-801, and [3H]AMPA.
In other binding studies aliquots of membrane suspension were
the experimental Rf values according to the formula Rm
)
log[(1/Rf) - 1]. Higher Rm values indicate higher lipophilicity.
Testin g of An ticon vu lsa n t Activity. Au d iogen ic Sei-
zu r es in DBA/2 Mice. All experiments were performed with
DBA/2 mice which are genetically susceptible to sound-induced
seizures.23 DBA/2 mice (8-12 g; 22-25 days old) were
purchased from Charles River (Calco, Como, Italy). Groups
of 10 mice of either sex were exposed to auditory stimulation
30 min following administration of vehicle or each dose of
drugs studied. The compounds were given ip (0.1 mL/10 g of
body weight of the mouse) as a freshly prepared solution in
50% dimethyl sulfoxide (DMSO) and 50% sterile saline (0.9%
NaCl). Individual mice were placed under a hemispheric
Perspex dome (diameter 58 cm) and were allowed 60 s for
habituation. Assessment of locomotor activity was also made
during this time period. Auditory stimulation (12-16 kHz,
109 dB) was applied for 60 s or until tonic extension occurred
and induced a sequential seizure response in control DBA/2
mice, consisting of an early wild running phase, followed by
generalized myoclonus and tonic flexion and extension some-
times followed by respiratory arrest. The control and drug-
treated mice were scored for latency to and incidence of the
different phases of the seizures.24 The time course of the
anticonvulsant action of 2a , 3a , and 1 was determined follow-
ing the administration of 33 µmol/kg of each benzodiazepine
derivative to groups of 10 mice that were tested for sound-
induced seizure responses at 5-240 min after drug adminis-
tration.

3,5-Dihydro-4H-2,3-benzodiazepine-4-thiones
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3415
incubated at the appropriate temperature for 60 min with 50
nM [3H]CNQX (specific activity, 18.3 Ci/mmol), 10 nM [3H]-
5,7-DCKA (specific activity, 18.3 Ci/mmol), or [3H]ACPD
(mixture of [3H](1S,3R)ACPD and [3H](1R,3S)ACPD, specific
activity 30-50 Ci/mmol). Nonspecific binding was defined as
the binding measured in the presence of 0.2 mM quisqualate,
0.1 mM d-serine, or 0.2 mM (1S,3R)ACPD, respectively.
Refer en ces
(1) De Sarro, G.; Chimirri, A.; De Sarro, A.; Gitto, R.; Grasso, S.;
Giusti, P.; Chapman, A. G. GYKI 52466 and Related 2,3-
Benzodiazepines as Anticonvulsant Agents in DBA/2 Mice. Eur.
J . Pharmacol. 1995, 294, 411-422.
(2) Chimirri, A.; De Sarro, G.; De Sarro, A.; Gitto, R.; Grasso, S.;
Quartarone, S.; Zappala`, M.; Giusti, P.; Libri, V.; Constanti, A.;
Chapman, A. G. 1-Aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-
ones: Novel AMPA Receptor Antagonists. J . Med. Chem. 1997,
40, 1258-1269.
Effects on Motor Movem en ts. Male Swiss mice (20-26
g, 48-54 days old) were purchased from Charles River (Calco,
Como, Italy). Groups of 10 mice were trained to do coordinated
motor movements continuously for 2 min on a rotarod, 3 cm
diameter, at 8 rpm (U. Basile, Comerio, Varese, Italy).
Impairment of coordinated motor movements was defined as
the inability of the mice to remain on the rotarod for a 2-min
test period.29 The ability of the mice to remain on the rotarod
was tested 30 min after administration of various compounds.
(3) Monaghan, D. T., Wenthold, R. J., Eds.; The Ionotropic Glutamate
Receptors; Humana Press: New J ersey, 1997.
(4) Krogsgaard-Larsen, P.; Ebert, B.; Lund, T. M.; Bra¨uner-Osborne,
H.; Sløk, F. A.; J ohansen, T. N.; Brehm, L.; Madsen, U. Design
of Excitatory Amino Acid Receptor Agonists, Partial Agonists
and Antagonists: Ibotenic Acid as a Key Lead Structure. Eur.
J . Med. Chem. 1996, 31, 515-537.
(5) Lin, Z.; Kadaba, P. K. Molecular Targets for the Rational Design
of Antiepileptic Drugs and Related Neuroprotective Agents. Med.
Res. Rev. 1997, 17, 537-572.
Electr op h ysiology. Transverse slices of olfactory cortex
(∼450 µm thick) were obtained from adult guinea pigs (250-
400 g, either sex), as previously described,30 and stored in
oxygenated Krebs solution at 32 °C for at least 30 min before
being transferred to an immersion chamber for recordings. The
composition of the Krebs fluid was (mM) as follows: NaCl, 118;
KCl, 3; CaCl2, 1.5; NaHCO3, 25; MgCl2‚6H2O, 1; and D-glucose,
11 (bubbled with 95% O2:5% CO2, pH 7.4). Conventional
intracellular recordings were obtained from the periamygda-
loid area of the slices within the olfactory pyramidal cell layer
II-III,31 using glass microelectrodes (tip resistances 40-60
MΩ) filled with 4 M potassium acetate. Voltage clamp
recordings were made at a holding membrane potential of -80
mV with the aid of a DAGAN 8100 sample-and-hold voltage
clamp preamplifier (2-3 kHz switching frequency, 25% duty
cycle). Sampled membrane currents (filtered at 30 Hz, low
pass) and voltage were recorded on a Gould 2400 ink jet chart
recorder. The following compounds were tested: AMPA, 3i,
and 1. In addition, slices were continuously superfused with
1 µM TTX to block voltage-activated sodium currents and
underlying repetitive firing at the peak of AMPA responses.
AMPA and TTX were freshly prepared in Krebs solution,
whereas compounds 3i and 1 were predissolved in dimethyl
sulfoxide (DMSO) to give 10 mM stock solutions and subse-
quently diluted in Krebs solution (containing 0.1-1% v/v
DMSO) immediately prior to use. These concentrations of
DMSO had no deleterious effects on neuronal membrane
properties or AMPA-induced inward currents. All measure-
ments were performed before, during, and after bath applica-
tion of pharmacological agents so that each neuron served as
its own control.
(6) (a) Ohmori, J .; Sakamoto, S.; Kubota, H.; Shimizu-Sasamata,
M.; Okada, M.; Kawasaki, S.; Nikada, K.; Togami, J .; Furuya,
T.; Murase, K. 6-(1H-Imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxa-
linedione Hydrochloride (YM90K) and Related Compounds:
Structure-Activity Relationships for the AMPA-Type Non-
NMDA Receptor. J . Med. Chem. 1994, 37, 467-475. (b) Desos,
P.; Lespagnol, J . M.; Morain, P.; Lestage, P.; Cordi, A. A.
Structure-Activity Relationships in a Series of 2(1H)-Quinolones
Bearing Different Acidic Function in the 3-Position: 6,7-Dichloro-
2(1H)-oxoquinoline-3-phosphonic Acid, a New Potent and Selec-
tive AMPA/Kainate Antagonist with Neuroprotective Properties.
J . Med. Chem. 1996, 39, 197-206. (c) Honore´, T.; Davies, S. N.;
Drejer, J .; Fletcher, E. J .; J acobsen, P.; Lodge, D.; Nielsen, F.
E. Quinoxalinediones: Potent Competitive non-NMDA Glutamate
Receptor Antagonists. Science 1988, 241, 701-707. (d) Honore´,
T.; Drejer, J .; Nielsen, F. E.; Nielsen, M. Non-NMDA Glutamate
Receptor Antagonist 3H-CNQX Binds with Equal Affinity to two
Agonist States of Quisqualate Receptor. Biochem. Pharmacol.
1989, 38, 3207-3212. (e) Cai, S. X.; Huang, J . C.; Espitia, S. A.;
Tran, M.; Ilyin, V. I.; Hawkinson. J . E.; Woodward, R. M.; Weber,
E.; Keana, J . F. W. 5-(N-Oxyaza)-7-substituted-1,4-dihhydro-
quinoxaline-2,3-diones: Novel, Systemically Active and Broad
Spectrum Antagonists for NMDA/glycine, AMPA and Kainate
Receptors. J . Med. Chem. 1997, 40, 3679-3686.
(7) De Sarro, G. B.; Ongini, E.; Bertorelli, R.; Aguglia, U.; De Sarro,
A. Excitatory Amino Acid Neurotransmission through both
NMDA and Non-NMDA Receptors is Involved in the Anticon-
vulsant Activity of Felbamate in DBA/2 Mice. Eur. J . Pharmacol.
1994, 262, 11-19.
(8) Pelletier, J . C.; Hesson, D. P.; J ones, K. A.; Costa, A. M.
Substituted 1,2-Dihydrophthalazines: Potent, Selective, and
Noncompetitive Inhibitors of the AMPA Receptor. J . Med. Chem.
1996, 39, 343-346.
(9) (a) Ornstein, P. L.; Arnold, M. B.; Augenstein, N. K.; Lodge, D.;
Leander, J . D.; Schoepp, D. D. (3SR,4aRS,6RS,8aRS)-6-[2-(1H-
Tetrazol-5-yl)ethyl]decahydro-isoquinoline-3-carboxylic Acid: A
Structurally Novel, Systemically Active, Competitive AMPA
Receptor Antagonist. J . Med. Chem. 1993, 36, 6, 2046-2048.
(b) Ornstein, P. L.; Arnold, M. B.; Allen, N. K.; Leander, J . D.;
Tizzano, J . P.; Lodge, D.; Schoepp, D. D. (3SR,4aRS,6RS,8aRS)-
6-(1H-Tetrazol-5-yl)decahydro-isoquinoline-3-carboxylic Acid, A
Novel Competitive, Systemically Active NMDA and AMPA
Receptor Antagonist. J . Med. Chem. 1995, 38, 4885-4890.
(10) (a) Chapman, A. G.; Smith, S. E.; Meldrum, B. S. The Anticon-
vulsant Effect of the Non-NMDA Antagonists, NBQX and GYKI
52466 in Mice. Epilepsy Res. 1991, 9, 92-96. (b) Smith, S. E.;
Durmuller, N.; Meldrum, B. S. The non-N-methyl-D-aspartate
Receptor Antagonists, GYKI 52466 and NBQX are Anticonvul-
sant in two Animal Models of Reflex Epilepsy. Eur. J . Pharma-
col. 1991, 201, 179-183. (c) Chapman, A. G.; Al-Zubaidy, Z.;
Meldrum, B. S. Aniracetam Reverses the Anticonvulsant Action
of NBQX and GYKI 52466 in DBA/2 Mice. Eur. J . Pharmacol.
1993, 231, 301-303. (d) Donevan, S. D.; Rogawski, M. A. GYKI
52466, a 2,3-Benzodiazepine, is a Highly Selective, Noncompeti-
tive Antagonist of AMPA/Kainate Receptor Responses. Neuron
1993, 10, 51-59.
(11) (a) Rogawski, M. A. Therapeutic Potential of Excitatory Amino
Acid Antagonists: Channel Blockers and 2,3-Benzodiazepines.
Trends Pharmacol. Sci. 1993, 14, 325-331. (b) Zorumski, C. F.;
Yamada, K. A.; Price, M. D.; Olney J . W. Benzodiazepine
Recognition Side Associated with non-NMDA Glutamate Recep-
tor. Neuron 1993, 10, 61-67. (c) Donevan, S. D.; Yamaguchi, S.
I.; Rogawski, M. A. Non-N-methyl-D-aspartate Receptor Antago-
nism by 3-N-Substituted 2,3-Benzodiazepines: Relationship to
Anticonvulsant Activity. J . Pharmacol. Exp. Ther. 1994, 271,
25-29.
Sta tistica l An a lysis. Statistical comparisons between
groups of control and drug-treated animals were made using
Fisher’s exact probability test (incidence of the seizure phases)
or ANOVA followed by post hoc Dunnett’s t-test (rectal
temperatures). The ED50 values of each phase of the audio-
genic seizure or seizures induced by electroshock or pentyle-
neterazole was determined for each dose of compound admin-
istered, and dose-response curves were fitted using a computer
program by the method of Litchfield and Wilcoxon.32 The
relative anticonvulsant activities were determined by com-
parison of respective ED50 values. The dose which induced
50% of mice to fall from the rotarod (TD50 values) was
estimated using the method of Litchfield and Wilcoxon.32 The
relative activities were determined by comparison of respective
TD50 values. For the binding experiments IC50 values for the
displacement of [3H]flumazenil or of other ligands were
determined by the nonlinear curve-fitting program based on
Ligand.33 All data of electrophysiology are expressed as mean
( standard error of the mean (SEM). Statistical significance
between control and test groups of data means was tested
using a two-tailed Student’s t test.
Ack n ow led gm en t. Financial support from CNR is
gratefully acknowledged.

3416 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Chimirri et al.
(12) (a) Lodge, D.; Bond, A.; Birds, D.; Palmer, A. J . Interaction
between 2,3-Benzodiazepine and Cyclothiazide on the AMPA
Subtype of Glutamate Receptor. Neurosci. Abs. 1993, 19, 124.4.
(b) Rammers, G.; Swandulla, D.; Collingridge, G. L.; Hartman,
S.; Passons, C. G. Interaction of 2,3-Benzodiazepine and Cy-
clothiazide at AMPA Receptor: Patch Clamp Recordings in
Cultured Neurones and Area CA1 in Hippocampal Slices. Br.
J . Pharmacol. 1996, 117, 1209-1221.
(13) (a) Lodge, D.; Bond, A.; O’Neill, M.; Hicks, C. A.; J ones, M. G.
Stereoselective Effects of 2,3-Benzodiazepines In Vivo: Electro-
physiology and Neuroprotection Studies. Neuropharmacology
1996, 35, 1681-1688. (b) Bleakman, D.; Ballyk, B. A.; Schoepp,
D. D.; Palmer, A. J .; Bath, C. P.; Sharpe, E. F.; Wooley, M. L.;
Bufton, H. R.; Kamboj, R. K.; Tarnawa, I.; Lodge, D. Activity of
2,3-Benzodiazepines at Native Rat and Recombinant Human
Glutamate Receptors In Vitro: Stereospecificity and Selectivity
Profiles. Neuropharmacology 1996, 35, 1689-1702.
102, 551-552. (b) Bliss, T. V. P.; Collingridge G. L. A Synaptic
Model of Memory: Long-Term Potentiation in the Hippocampus.
Nature 1993, 361, 31-39. (c) Pasternain A. V.; Morales, M.;
Lerma, J . Selective Antagonism of AMPA Receptors Unmasks
Kainate Receptor-Mediate Responses in Hippocampal Neurones.
Neuron 1995, 14, 185-189.
(22) (a) Boyce, C. B. C.; Milborrow, B. V. A Simple Assessment of
Partition Data for Correlating Structure and Biological Activity
Using Thin-Layer Chromatography. Nature 1965, 208, 537-539.
(b) Pietrogrande, M. C.; Borea, P. A.; Biagi, G. L. Lipophilicity
Measurements of Benzodiazepine-Receptor Ligands by Reversed-
Phase Liquid Chromatography. Comparison between High-
Performance Liquid and Thin-Layer Chromatography. J . Chro-
matogr. 1988, 447, 404-409.
(23) (a) Hall, C. S. Genetic Differences in Fatal Audiogenic Seizures
between two Inbred Strains of House Mice. J . Hered. 1947, 38,
2-6. (b) Collins, R. L. Audiogenic Seizures. In Experimental
Models of Epilepsy; Purpura, P., Penry, J . K, Tower, D.,
Woodbury, D. M., Walter, R., Eds.; Raven Press: New York,
1972; pp 347-372.
(14) Tarnawa, I.; Farkas, S.; Berzsenyi, P.; Pataki, A.; Andrasi, F.
Electrophysiological Studies with a 2,3-Benzodiazepine Muscle
Relaxant: GYKI 52466. Eur. J . Pharmacol. 1989, 167, 193-
199.
(15) (a) Chimirri, A.; De Sarro, A.; De Sarro, G. B.; Grasso, S.;
Trimarchi, G. R.; Zappala`, M. Synthesis and Anticonvulsant
Properties of 2,3,3a,4-Tetrahydro-1H-pyrrolo[1,2-a]benzimidazol-
1-ones. J . Med. Chem. 1989, 32, 93-95. (b) Chimirri, A.; Grasso,
S.; Monforte, A. M.; Zappala`, M.; De Sarro, A.; De Sarro, G. B.
Synthesis and Anticonvulsant Properties of 3-(1,3,4-Thiadiazol-
2-yl)thiazolidin-4-ones. Farmaco 1991, 46, 935-943. (c) De
Sarro, A.; De Sarro, G. B.; Chimirri, A.; Grasso, S.; Monforte,
A. M.; Zappala`, M. Anticonvulsant Activity of Pyrrolo[1′,2′:1,2]-
imidazo[4,5-b]pyridines, Pyrrolo[2′,1′:2,3]imidazo[4,5-c]pyridines
and Pyrrolo[2,1-f]purines in DBA/2 Mice. Gen. Pharmacol. 1994,
25, 1027-1031.
(16) (a) De Sarro, G. B.; Zappala`, M.; Grasso, S.; Chimirri, A.;
Spagnolo, C.; De Sarro, A. Molecular Requirement for Anticon-
vulsant Activity in a Series of 1,5-Benzodiazepines and [1,2,4]-
Oxadiazolo[4,5-a][1,5]benzodiazepine Derivatives. Mol. Neuro-
pharmacol. 1992, 1, 195-202. (b) De Sarro, G.; Chimirri, A.; De
Sarro, A.; Gitto, R.; Grasso, S.; Zappala`, M. 5H-[1,2,4]Oxadiazolo-
[5,4-d][1,5]benzothiazepines as Anticonvulsant Agents in DBA/2
Mice. Eur. J . Med. Chem. 1995, 30, 925-929.
(24) De Sarro, G. B.; Croucher, M. J .; Meldrum, B. S. Anticonvulsant
Actions of DS 103-282: Pharmacological Studies in Rodents and
the Baboon Papio papio. Neuropharmacology 1984, 23, 526-
530.
(25) Swinyard, E. A.; Brown, W. C.; Goodman, L. S. Comparative
Assays of Antiepileptic in Mice and Rats. J . Pharmacol. Exp.
Ther. 1952, 106, 319-330.
(26) Swinyard, E. A.; Woodhead, J . M. Experimental Detection,
Quantification and Evaluation of Anticonvulsants. In Epileptic
Drugs; Woodbury, D. M., Penry, J . K., Pippenger, C. E., Eds.;
Raven Press: New York, 1982; pp 111-126.
(27) De Sarro, G. B.; Ongini, E.; Bertorelli, R.; Aguglia, U.; De Sarro,
A. Excitatory Amino Acid Neurotransmission through both
NMDA and Non-NMDA Receptors is Involved in the Anticon-
vulsant Activity of Felbamate in DBA/2 mice. Eur. J . Pharmacol.
1994, 262, 11-19.
(28) (a) Apud, J . A.; Ito Y. Semi-preparative Purification of an
Endogenous Ligand for Brain Serotonin-2-receptor by Coil
Planet Centrifuge Counter-Current Chromatography. J . Chro-
matogr. 1991, 538, 177-185. (b) Bylund, D. B.; Yamamura H.
I. Methods for Receptor Binding. In Methods in Neurotransmitter
Receptor Analysis; Yamamura, H. I., Enna, S. J ., Kuhar, M. J .,
Eds.; Raven Press: New York, 1990; pp 1-35. (c) Murphy, D.
E.; Schneider, J .; Boehm, C.; Lehman J .; Williams, M. Binding
of [3H]3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid to
(17) (a) De Sarro, G. B.; Zappala`, M.; Grasso, S.; Chimirri, A.; De
Sarro, A. Molecular Requirement for Anticonvulsant Activity in
a Series of Thiazolo-1,4-benzodiazepines and Comparison with
Classical Benzodiazepines. Gen. Pharmacol. 1993, 24, 877-884.
(b) De Sarro, G. B.; Chimirri, A.; Zappala`, M.; Giusti, P.;
Lipartiti, M.; De Sarro, A. Azirino[1,2-d][1,4]benzodiazepine
Derivatives and Related 1,4-Benzodiazepines as Anticonvulsant
Agents in DBA/2 Mice. Gen. Pharmacol. 1996, 27, 1155-1162.
(18) (a) Chapman, A. G.; Croucher, M. J .; Meldrum, B. S. Evaluation
of Anticonvulsant Drugs in DBA/2 Mice with Sound-Induced
Seizures. Arzneim.-Forsch. 1984, 34, 1261-1264. (b) Seyfried,
T. N.; Glaser, G. H. A Review of Mouse Mutants as Genetic
Models of Epilepsy. Epilepsia 1985, 26, 143-150. (c) Engstrom,
F. L.; Woodbury, D. M. Seizure Susceptibility in DBA/2 and C57
Mice: the Effects of Various Convulsants. Epilepsia 1988, 29,
389-395.
Rat Brain Membrane:
a Selective High-Affinity Ligand for
N-Methyl-D-aspartate Receptors J . Pharmacol. Exp. Ther. 1987,
240, 778-784. (d) Subramaniam, S.; McGonigle P. Quantitative
Autoradiographic Characterization of the Binding of (+)-5-
Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]-
MK-801) in Rat Brain J . Pharmacol. Exp. Ther. 1991, 256, 811-
819. (e) Nielsen, E. O.; Drejer, J .; Cha, J . H.; Young, A. B.;
Honore´, T. Autoradiographic Characterization and Localization
of Quisqualate Binding Sites in Rat Brain using the Antagonists
[3H]6-Cyano-7-nitroquinoxaline-2,3-dione: Comparison with [3H]-
(R,S)-R-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Bind-
ing Sites. J . Neurochem. 1990, 54, 686-695.
(19) Ito, I.; Tanabe, S.; Kohda, A.; Sugiyama, H. Allosteric Potentia-
tion of Quisqualate Receptors by Nootropic Drug Aniracetam.
J . Physiol. (London) 1990, 424, 533-543.
(20) (a) Chapman, A. G.; Meldrum, B. S. Noncompetitive N-methyl-
D-aspartate Antagonists Protect Against Sound-Induced Seizures
in DBA/2 Mice. Eur. J . Pharmacol. 1989, 166, 201-211. (b) De
Sarro, G. B.; De Sarro, A. Anticonvulsant Activity of Competitive
Antagonists of NMDA Receptor in Genetically Epilepsy Prone
Rats. Eur. J . Pharmacol. 1992, 215, 221-229. (c) De Sarro, G.
B.; De Sarro, A. Anticonvulsant Properties of Non-Competitive
Antagonists of NMDA Receptor in Genetically Epilepsy Prone
Rats. Comparison with CPPene. Neuropharmacology 1993, 32,
51-58. (d) McEntee, W. C.; Crook, T. H. Glutamate, its Role in
Learning, Memory, and the Aging Brain. Psychopharmacology
1993, 111, 391-401. (e) Lo¨scher, W. R.; Annies R.; Ho¨nack, D.
Comparison of Competitive and Uncompetitive NMDA Receptor
Antagonist with Regard to Monoaminergic Neuronal Activity
and Behavioural Effects in Rats. Eur. J . Pharmacol. 1993, 242,
263-274.
(29) Dunham, N. W.; Miya, T. S. A Note on a Simple Apparatus for
Detecting Neurological Deficit in Rats and Mice. J . Am. Pharm.
Assoc. 1957, 46, 208-209.
(30) Constanti, A.; Bagetta, G.; Libri, V. Persistent Muscarinic
Excitation in Guinea-Pig Olfactory Cortex Neurons: Involve-
ment of a Slow Post-Stimulus After Depolarizing Current.
Neuroscience 1993, 56, 887-904.
(31) Libri, V.; Constanti, A.; Calaminici, M.; Nistico`, G. A Comparison
of the Muscarinic Response and Morphological Properties of
Identified Cells in the Guinea-pig Olfactory Cortex in vitro.
Neuroscience 1994, 59, 331-347.
(32) Litchfield, J . T.; Wilcoxon, F. A Simplified Method of Evaluating
Dose-Effects Experiments. J . Pharmacol. Exp. Ther. 1949, 96,
99-113.
(33) McPherson, G. I. Ligand (release 2.0), Elsevier Biosoft, Cam-
bridge, 1987.
(21) (a) Chiamulera, C.; Costa S.; Reggiani, A. Effect of NMDA- and
Strychnine- Insensitive Glycine Site Antagonists on NMDA-
Mediated Convulsions and Learning, Psychopharmacology 1990,
J M9800393