1-aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones: Novel AMPA receptor antagonists

Article abstract of DOI:10.1021/jm960506l

Our previous publication (Eur. J. Pharmacol. 1995, 294, 411-422) reported preliminary chemical and biological studies of some 2,3- benzodiazepines, analogues of 1-(4-aminophenyl)-4-methyl-7,8- (methylenedioxy)-5H-2,3-benzodiazepine (1, GYKI 52466), which have been shown to possess significant anticonvulsant activity. This paper describes the synthesis of new 1-aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones and the evaluation of their anticonvulsant effects. The observed findings extend the structure-activity relationships previously suggested for this class of anticonvulsants. The seizures were evoked both by means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. 1-(4'-Aminophenyl)- (38) and 1-(3'-aminophenyl)-3,5-dihydro- 7,8-dimethoxy-4H-2,3-benzodiazepin-4-one (39), the most active compounds of the series, proved to be more potent than 1 in all tests employed. In particular, the ED₅₀ values against tonus evoked by auditory stimulation were 12.6 μmol/kg for derivative 38, 18.3 μmol/kg for 39, and 25.3 μmol/kg for 1. Higher doses were necessary to block tonic extension induced both by maximal electroshock and by pentylenetetrazole. In addition these compounds exhibited anticonvulsant properties that were longer lasting than those of compound 1 and were less toxic. The novel 2,3-benzodiazepines were also investigated for a possible correlation between their anticonvulsant activities against convulsions induced by 2-amino-3-(3-hydroxy-5- methylisoxazol-4-yl)propionic acid (AMPA) and their affinities for benzodiazepine receptors (BZR). The 2,3-benzodiazepines did not affect the binding of [³H]flumazenil to BZR, and conversely, their anticonvulsant effects were not reversed by flumazenil. On the other hand the 2,3- benzodiazepines antagonized seizures induced by AMPA and aniracetam in agreement with an involvement of the AMPA receptor. In addition, both the derivative 38 and the compound 1 markedly reduced the AMPA receptor-mediated membrane currents in guinea-pig olfactory cortical neurons in vitro in a noncompetitive manner. The derivatives 25 and 38-40 failed to displace specific ligands from N-methyl-D-aspartate (NMDA), AMPA/kainate, or metabotropic glutamate receptors.

Full text of DOI:10.1021/jm960506l

1258
J . Med. Chem. 1997, 40, 1258-1269
1-Ar yl-3,5-d ih yd r o-4H-2,3-ben zod ia zep in -4-on es: Novel AMP A Recep tor
An ta gon ists
Alba Chimirri,*,† Giovambattista De Sarro,‡ Angela De Sarro,§ Rosaria Gitto,† Silvana Grasso,†
Silvana Quartarone,† Maria Zappala`,† Piero Giusti, Vincenzo Libri,|,# Andrew Constanti,# and
Astrid G. Chapman∇
Dipartimento Farmaco-Chimico, Universita` di Messina, Viale Annunziata, 98168 Messina, Italy, Dipartimento di Medicina
Sperimentale e Clinica, Universita` di Reggio Calabria, Istituto di Farmacologia, Facolta` di Medicina, Universita` di Messina,
Dipartimento di Farmacologia, Universita` di Padova, Dipartimento di Biologia, Universita` di Roma “Tor Vergata”, Italy,
Department of Pharmacology, School of Pharmacy, University of London, WC1N 1AX, U.K., and Department of Neurology,
Institute of Psychiatry, University of London, SE5 8AF, U.K.
Received J uly 15, 1996X
Our previous publication (Eur. J . Pharmacol. 1995, 294, 411-422) reported preliminary
chemical and biological studies of some 2,3-benzodiazepines, analogues of 1-(4-aminophenyl)-
4-methyl-7,8-(methylenedioxy)-5H-2,3-benzodiazepine (1, GYKI 52466), which have been shown
to possess significant anticonvulsant activity. This paper describes the synthesis of new 1-aryl-
3,5-dihydro-4H-2,3-benzodiazepin-4-ones and the evaluation of their anticonvulsant effects.
The observed findings extend the structure-activity relationships previously suggested for
this class of anticonvulsants. The seizures were evoked both by means of auditory stimulation
in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. 1-(4′-
Aminophenyl)- (38) and 1-(3′-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-
4-one (39), the most active compounds of the series, proved to be more potent than 1 in all
tests employed. In particular, the ED50 values against tonus evoked by auditory stimulation
were 12.6 µmol/kg for derivative 38, 18.3 µmol/kg for 39, and 25.3 µmol/kg for 1. Higher doses
were necessary to block tonic extension induced both by maximal electroshock and by
pentylenetetrazole. In addition these compounds exhibited anticonvulsant properties that were
longer lasting than those of compound 1 and were less toxic. The novel 2,3-benzodiazepines
were also investigated for a possible correlation between their anticonvulsant activities against
convulsions induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and
their affinities for benzodiazepine receptors (BZR). The 2,3-benzodiazepines did not affect the
binding of [3H]flumazenil to BZR, and conversely, their anticonvulsant effects were not reversed
by flumazenil. On the other hand the 2,3-benzodiazepines antagonized seizures induced by
AMPA and aniracetam in agreement with an involvement of the AMPA receptor. In addition,
both the derivative 38 and the compound 1 markedly reduced the AMPA receptor-mediated
membrane currents in guinea-pig olfactory cortical neurons in vitro in a noncompetitive manner.
The derivatives 25 and 38-40 failed to displace specific ligands from N-methyl-D-aspartate
(NMDA), AMPA/kainate, or metabotropic glutamate receptors.
In tr od u ction
D-aspartic acid (NMDA) or 2-amino-3-(3-hydroxy-5-
methylisoxazol-4-yl)propionic acid (AMPA) and kainic
acid (non-NMDA) and the metabotropic receptors acti-
vated by trans-1-aminocyclopentane-1,3-dicarboxylic acid
(ACPD).3 Historically, NMDA receptor antagonists
have received intense interest as therapeutic agents,4
but the initial enthusiasm has been dampened by
increasing awareness of their various toxicities.5 Today,
there is considerable interest in the development of
potent and selective non-NMDA receptor antagonists
because of their important role in the treatment of
epilepsy as well as cerebral ischaemia and various forms
of neurodegenerative diseases.6,7 Studies utilizing po-
tent AMPA/kainate receptor antagonists such as 6-nitro-
7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) have
supplied experimental evidence that the design and the
synthesis of AMPA receptor antagonists could help to
elucidate the understanding of EAA receptor function
in the pathogenesis and propagation of epileptic phe-
nomena.
A large number of people worldwide suffer from
epilepsy, and at least 25% of them have seizures that
are resistant to available medical therapies.2 Treat-
ment of uncontrolled seizures therefore poses a continu-
ing challenge in epilepsy management. Existing anti-
epileptic drugs and those in current development prevent
seizures by a variety of mechanisms which include
modulation of sodium channels or thalamic calcium
currents, enhancement of GABA-mediated inhibition,
or antagonist action at excitatory amino acid (EAA)
receptors.
Much of the available evidences demonstrate that
EAA receptor antagonists are potent anticonvulsant
compounds. The more well-defined EAA receptors are
the ionotropic channels activated by either N-methyl-
* Author to whom correspondence should be addressed.
† Dipartimento Farmaco-Chimico.
‡ Dipartimento di Medicina Sperimentale e Clinica.
§ Istituto di Farmacologia.
Dipartimento di Farmacologia.
| Dipartimento di Biologia.
#
Department of Pharmacology.
It was previously reported8 that 1-(4-aminophenyl)-
4-methyl-7,8-(methylenedioxy)-5H-2,3-benzodiazepine (1,
∇ Department of Neurology.
X Abstract published in Advance ACS Abstracts, March 1, 1997.
S0022-2623(96)00506-7 CCC: $14.00 © 1997 American Chemical Society

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1259
Sch em e 1a
GYKI 52466) differs pharmacologically from conven-
tional 1,4-benzodiazepines, such as diazepam, in that
it lacks sedative-hypnotic activity and does not bind
to benzodiazepine receptors (BZR). Compound 1 shows
anticonvulsant and muscle-relaxant properties and
proved to be a highly selective AMPA/kainate receptor
antagonist; it does not affect NMDA, metabotropic
glutamate, or GABAA receptor-mediated responses.
With these considerations in mind and as a part of our
continuing studies on potential anticonvulsant agents9
(particularly benzodiazepine10 and benzothiazepine11
derivatives), we have recently reported1 the synthesis
and the anticonvulsant activity of some 2,3-benzodiaz-
epine derivatives that are analogues of 1. On the basis
of the significant AMPA receptor antagonist activity of
some of the compounds and in an attempt to increase
their potency and selectivity and improve the definition
of their structure-activity relationships (SAR), we now
report the synthesis and anticonvulsant activity of
several novel 1-aryl-3,5-dihydro-4H-2,3-benzodiazepin-
4-one derivatives. SAR of variation of N-3, C-8, and C-1
aromatic ring substitutions and the resulting modula-
tion of anticonvulsant activity are described. Com-
pounds were studied after intraperitoneal (ip) admin-
istration in DBA/2 mice, a strain genetically susceptible
to sound-induced seizures, which has been considered
an excellent animal model for generalized epilepsy and
for screening new anticonvulsant drugs.12 2,3-Benzo-
diazepines were also examined for anticonvulsant activ-
ity both against pentylenetetrazole and maximal elec-
troshock-induced seizures. In addition, we attempted
to correlate the anticonvulsant efficacies of some of the
2,3-benzodiazepines with their respective affinities for
the BZR or AMPA receptors. BZR affinities were
assessed by the potencies of the 2,3-benzodiazepines 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.
Since 1 and related compounds are known not to bind
competitively at the AMPA receptor,13 the AMPA af-
finities of 2,3-benzodiazepines were assessed indirectly
through their inhibitory action on AMPA-induced sei-
zures. The abilities of aniracetam, a compound which
potentiates the effects of AMPA,14 to reverse the inhibi-
tion of AMPA-induced seizures by 2,3-benzodiazepines
were also assessed. The results obtained were com-
pared to the findings previously reported1 for com-
pounds 24-26 and 32. Finally, we also conducted
electrophysiological experiments on guinea pig cortical
brain slice neurons to investigate the mode of inhibitory
action of some 2,3-benzodiazepines at AMPA receptor
sites.
a
Reagents: (a) HCl(g)-saturated dioxane, reflux, 1 h; (b) 35%
H2SO4, CrO3, acetone.
Sch em e 2a
a
Reagents: (a) NH2NH2‚H2O or MeNHNH2, EtOH, reflux, 3-4
h.
outlined in Schemes 1-4. Derivatives 27-30 and 34-
44 are new compounds, whereas the synthesis of 24-
26 and 31-33 was previously reported by Gatta et al.15
3-Methoxy- (2) or 3,4-dimethoxyphenethyl alcohol (3)
was condensed with aromatic aldehydes 4-9 to afford
1-arylisochromans 10-16. Derivatives 10-16 were
oxidized to 2-aroylphenylacetic acids 17-23 which, by
treatment with hydrazine or monomethylhydrazine,
Ch em istr y
1-Aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones 24-
44 were obtained through the synthetic procedures

1260 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Chimirri et al.
Sch em e 3a
over, N-acetyl derivatives 42-44 were obtained from the
analogous 2,3-benzodiazepin-4-ones 25, 28, and 38 by
treatment with an excess of acetic anhydride. The
structures of all the newly synthesized compounds were
determined by analytical and spectral data (1H NMR)
and are reported in the Experimental Section.
Lip op h ilicity Mea su r em en ts
The relative lipophilicity (Rm) (see the Experimental
Section) of the 2,3-benzodiazepines studied are sum-
marized in Table 1. As expected, the most lipophilic
compounds were 33 and 34, owing to the simulataneous
presence of a methyl group and a halogen atom.
Biologica l Resu lts
An ticon vu lsa n t Activity a ga in st Au d iogen ic Sei-
zu r es in DBA/2 Mice. The anticonvulsant properties
of compounds 24-44 were evaluated 30 min after ip
administration of several doses (dose range 3.3-200
µmol/kg) in DBA/2 mice. Table 1 reports the median
effective dose (ED50) values required to prevent clonic
and tonic phases of sound-induced seizures. The rank
order of anticonvulsant potency was as follows: 38 >
39 > 25 > 1 > 41 > 32 > 33 > 40 > 44 > 34 > 24 > 26
> 42 > 27. The remaining compounds showed no
activity. The wild running phase was significantly
reduced after ip administration of 24-27, 31-33, 38-
41, and 44 at the highest doses tested.
a
Reagents: (a) granulated tin, 37% HCl, reflux, 1 h.
Sch em e 4a
Following ip administration of some active compounds
such as 25,1 32,1 38, 40, and 44, 33 µmol/kg, maximum
protection was observed from 30 to 90 min for compound
38 and from 45 to 90 min for the others with subsequent
return to control seizure response at 180 min for all
tested derivatives, as shown in Figure 1 for 38, 40, and
44. In contrast, 1, 33 µmol/kg, displayed the maximum
protection from 5 to 15 min followed by gradual return
to control seizure response between 30 and 90 min
(Figure 1).
a
Reagents: (a) Ac2O, Et3N, CHCl3, room temperature, 1-2 h.
gave 2,3-benzodiazepin-4-ones 24-30 and 3-methyl-2,3-
benzodiazepin-4-ones 31-37, respectively. When an
amino group is present on the phenyl ring at C-1 of the
isochroman, the oxidation process fails to give the
expected aroylphenylacetic acids. Thus, derivatives 38-
41 were prepared by reduction with Sn/HCl of the
corresponding nitro analogues 29-30 and 36-37. More-
An ticon vu lsan t P r oper ties again st Maxim al Elec-
tr osh ock in Sw iss Mice. As shown in Table 2, the
Ta ble 1. ED50 Values (95% Confidence Limits) of 24-44 and 1 against the Clonic and Tonic Phases of the Audiogenic Seizures in
DBA/2 Mice after 30 min Pretreatmenta and Relative Lipophilicity (Rm) of the Same 2,3-Benzodiazepines
ED50, µmol/kg ((95% confidence limits)
ED50, mg/kg ((95% confidence limits)
compd
dose range (µmol/kg)
clonic phase
tonic phase
clonic phase
tonic phase
Rm
24b
25b
26b
27
28
29
30
31
32b
33
34
35
36
37
38
39
40
41
42
43
44
1
33-200
10-100
33-200
10-133
10-120
10-120
10-120
33-133
10-100
33-66
10-100
10-120
10-120
10-120
3.3-100
10-66
75.5 (47.5-120)
33.9 (26.0-44.2)
102 (76.1-137)
110 (79.3-151)
64.8 (45.0-93.3)
31.8 (24.8-40.6)
75.3 (60.7-93.2)
82.5 (58.6-116)
20.0 (12.6-31.9)
10.0 (7.71-13.1)
33.7 (25.1-45.2)
41.2 (29.7-56.5)
17.2 (11.9-24.8)
9.41 (7.34-12.0)
24.8 (20.0-30.7)
30.7 (21.9-43.5)
-0.052
-0.012
0.017
0.052
>120
>120
>44
>44
-0.218
-0.096
-0.140
0.189
0.000
0.250
0.288
-0.031
0.110
>120
>120
117 (93.5-146)
>120
>120
>44
>44
>44
>44
99.1 (82.2-119)
32.8 (26.2-40.9)
27.8 (23.0-33.3)
37.8 (23.7-60.1)
41.2 (36.0-47.2)
63.0 (33-121)
26.7 (14.7-48.2)
38.6 (33.6-44.4)
38.0 (20.0-72.0)
11.7 (7.35-18.6)
14.2 (12.4-16.3)
24.5 (12.8-47.0)
8.28 (4.56-15.0)
13.3 (11.6-15.3)
14.8 (7.78-28.0)
>120
>120
>120
>120
>120
>120
>44
>44
>44
>44
>44
>44
0.052
15.0 (9.01-24.0)
12.6 (8.01-19.0)
4.66 (2.80-7.46)
3.92 (2.49-5.91)
-0.602
-0.525
-0.374
-0.327
-0.052
-0.065
-0.673
-0.298
19.3 (16.9-22.0)
50.2 (34.6-73.0)
36.8 (28.3-47.7)
101 (52.0-194)
18.3 (16.0-20.8)
43.7 (31.3-61.0)
30.6 (23.8-39.3)
72.1 (47.6-109)
6.00 (5.25-6.84)
16.3 (11.2-23.7)
11.9 (9.20-15.5)
33.3 (17.6-65.6)
5.69 (4.98-6.47)
14.2 (10.2-19.8)
9.94 (7.73-12.8)
24.4 (16.1-36.9)
10-100
10-66
10-100
10-120
10-100
3.3-66
>120
>120
>44
>44
56.8 (39.3-82.1)
35.8 (24.4-52.4)
43.9 (31.2-61.9)
25.3 (16-40.0)
24.8 (17.2-35.8)
10.5 (7.15-24.7)
19.2 (13.6-27.0)
7.41 (4.68-11.7)
a
b
All data were calculated according to the method of Litchfield and Wilcoxon.41 Reference 1.

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1261
Ta ble 3. ED50 Values (95% Confidence Limits) of Some
2,3-Benzodiazepines against the Clonic and Tonic Seizures
Induced by icv Injection of AMPA in DBA/2 Micea
ED50, µmol/kg ((95% confidence limits)
compd dose range
clonic phase
tonic phase
25b
32b
38
39
40
1
10-200
10-200
10-100
10-66
10-200
10-100
66.0 (45.9-94.9)
76.1 (47.5-122.1)
32.1 (23.2-44.3)
30.9 (23.9-39.9)
72.1 (47.5-109.4)
57.5 (43.5-76.0)
42.6 (26.4-68.8)
69.6 (44.1-110.1)
25.0 (16.5-30.0)
27.8 (21.5-35.9)
62.4 (44.7-87.3)
40.5 (26.3-60.8)
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
2,3-benzodiazepines. All data were calculated according to the
b
method of Litchfield and Wilcoxon.41 Reference 1.
F igu r e 1. Anticonvulsant effects of 38, 40, 44, and 1 (33 µmol/
kg ip) against audiogenic seizures in DBA/2 mice. The
ordinate shows seizure score; the abscissa shows the time after
intraperiotoneal administration of drug in min. Ten animals
were used for the determination of each point.
Ta ble 2. ED50 Values (95% Confidence Limits) of Various
2,3-Benzodiazepines against the Maximal Electroshock and
Pentyleneterazole-Induced Seizures in Swiss Mice after 45 min
Pretreatmenta
ED50, µmol/kg ((95% confidence limits)
compd dose range maximal electroshock pentylenetetrazole
24
25
26
27
28
29
30
32
36
38
40
1
33-200
10-100
33-200
10-150
10-150
10-150
10-150
10-150
10-150
10-100
10-150
10-100
73.4 (34.6-155)
35.8 (28.6-44.7)
115 (81.2-164)
123 (98.7-154)
98.2 (56.0-172)
68.2 (54.6-85.2)
145 (83.4-254)
>150
>150
>150
>150
>150
89.9 (51.5-157)
>150
>150
>150
42.7 (26.5-68.8)
>150
15.9 (7.3-33.5)
22.6 (11.7-43.8)
70.8 (44.7-112)
68.3 (56.2-83.1)
57.2 (41.5-78.8)
35.7 (29.3-43.4)
a
All data were calculated according to the method of Litchfield
and Wilcoxon.41
tonic extension of the seizures induced by maximal
electroshock was significantly reduced 45 min after ip
administration of 24-27, 32, 38, 40, and 1, while
compounds 28-30 and 36 at doses up to 150 µmol/kg
ip had no anticonvulsant activity against maximal
electroshock seizures.
An ticon vu lsa n t P r op er ties a ga in st P en tylen e-
tetr a zole-In d u ced Seizu r es in Sw iss Mice. As
shown in Table 2, the clonic phase of the seizures
induced by pentylenetetrazole was significantly reduced
45 min after ip administration of 24-26, 32, 38, 40, and
1 at doses higher than those which protected against
maximal electroshock. Compounds 27-30 and 36 at
doses up to 150 µmol/kg ip had no anticonvulsant
properties against seizures induced by pentylenetetra-
zole.
F igu r e 2. Anticonvulsant effects of 38-40 and 1 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.
tonic seizures (11.7 nmol AMPA icv). The clonic and
tonic phases of the seizure induced by AMPA were
significantly reduced 30 min after ip administration
both of 25 and 32 as already reported1 and of 38-40
and 1 as shown in Figure 2.
The highest doses of 2,3-benzodiazepines studied
induced ataxia, splayed hind limbs, and tremor in some
animals during the period of maximal anticonvulsant
activity.
P r etr ea tm en t w ith An ir a ceta m . The icv injection
of aniracetam alone (12.5, 50, and 100 nmol), 60 min
before testing, did not significantly modify the audio-
genic seizure response in DBA/2 mice, but aniracetam
(50 nmol icv), 60 min before testing, reversed the
anticonvulsant effects of 2,3-benzodiazepines in DBA/2
mice. Aniracetam reduced the anticonvulsant efficacy
of 25, 32, 38, 40, and 1 (Table 4), resulting in an increase
of anticonvulsant ED50 values ranging from 2.6- to 7.3-
The most active compounds of the series were further
evaluated, and the biological results are reported below.
An ticon vu lsa n t Activity a ga in st AMP A-In d u ced
Seizu r es in DBA/2 Mice. Intracerebroventricular (icv)
administration of 10 µL of AMPA (1-10 nmol) induced
generalized seizures in DBA/2 mice, with CD97 (the dose
which induced convulsions in 97% of mice) values of 9.7
nmol for all-limb clonic seizures (latency 1.3 ( 0.3 min)
and 11.7 nmol for forelimb tonic seizures (latency 1.9 (
0.4 min), respectively. Table 3 reports the anticonvul-
sant ED50 values of a series of 2,3-benzodiazepines
against AMPA-induced clonic (9.7 nmol AMPA icv) and

1262 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Chimirri et al.
Ta ble 5. ED50 Values (95% Confidence Limits) of Some
2,3-Benzodiazepines against the Clonic and Tonic Phases of the
Audiogenic Seizures after Concomitant Treatment with
Flumazenil in DBA/2 Micea
Ta ble 4. ED50 Values (95% Confidence Limits) of Some
2,3-Benzodiazepines against the Clonic and Tonic Phases of the
Audiogenic Seizures after Pretreatment with Aniracetam in
DBA/2 Micea
ED50, µmol/kg ((95% confidence limits)
ED50, µmol/kg ((95% confidence limits)
compd dose range
clonic phase
tonic phase
compd flumazenil, µmol
clonic phase
tonic phase
25b
32b
38
40
1
33-300
33-300
10-200
10-200
10-200
215 (142-324)*
259 (176-380)*
65.4 (44.5-96.2)*
141 (101-198)*
134 (88.8-203)*
157 (114-217)*
197 (137-281)*
58.2 (43.4-77.9)*
112 (83.8-150)*
100 (63.4-158)*
25b
38
39
40
1
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)
14.1 (10.1-19.9)
12.0 (6.82-21.0)
21.0 (17.2-25.8)
29.2 (8.90-48.7)
51.2 (24.8-106)
45.9 (22.5-93.7)
37.8 (23.7-60.1)
39.5 (29.6-53.7)
35.8 (26.2-49.0)
41.3 (28.0-60.7)
12.0 (6.82-21.0)
10.2 (7.61-13.7)
18.2 (8.90-48.2)
17.5 (8.70-35.1)
38.5 (21.3-69.6)
32.2 (17.7-58.8)
26.7 (14.7-48.2)
29.7 (20.9-42.1)
a
All data were calculated according to the method of Litchfield
and Wilcoxon.41 Significant differences between ED50 values of
group treated with aniracetam + 2,3-benzodiazepine and group
treated with 2,3-benzodiazepine alone (Table 1) are denoted *P <
24.72
b
0.01. Reference 1.
a
All data were calculated according to the method of Litchfield
and Wilcoxon.41 Reference 1.
b
fold. The corresponding aniracetam-induced shifts to
the right of the dose-response curves for protection by
38 and 40 against sound-induced clonic and tonic
seizures are shown in Figure 3.
The highest doses of some 2,3-benzodiazepines stud-
ied induced ataxia, splayed hind limbs, and tremor in
some animals during the period of maximal anticon-
vulsant activity.
Tr ea tm en t w ith F lu m a zen il. As previously dem-
onstrated,16 flumazenil, administered ip at 8.24 or 24.7
µmol/kg, is not itself convulsant and does not signifi-
cantly modify the phases of the audiogenic seizure
response in DBA/2 mice, but antagonizes the anticon-
vulsant action of classical 1,4-benzodiazepines. In order
to ascertain the possible involvement of benzodiazepine
receptors in the antiseizure activity of 2,3-benzodiaz-
epines, derivatives 25, 38-40, and 1 were administered
concomitantly with flumazenil, but no significant modi-
fication of the antiseizure effects of these derivatives
was observed. The ED50 values for the different phases
of audiogenic seizures are reported in Table 5.
In h ibition of Bin d in g, in Vitr o. The potency of the
2,3-benzodiazepines as inhibitors of [3H]flumazenil bind-
ing to membranes from cortex was evaluated, and no
inhibition was observed (IC50 > 10000 nM).
By using crude cortical synaptic membranes prepared
from the rat brain, the derivative 25 and 38-40 failed
to displace [3H]spiperone from dopamine and 5-hydrox-
ytryptamine1 receptors; [3H]ketanserin from 5-hydrox-
ytryptamine2 receptors; [125I]pindolol from â-adrenergic
F igu r e 3. Antagonism by aniracetam (50 nmol icv) of the anticonvulsant effects of 38 and 40 against audiogenic seizures 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.

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1263
Ta ble 6. ED50 and TD50 Values (95% Confidence Limits) of
Various 2,3-Benzodiazepines on Locomotion Assessed by
Rotarod Test following 30 min Pretreatmenta
lar type and degree of depression was observed in the
presence of 50 µM 1 (Figure 4). In each case, the
depression of 1, 2, and 5 µM AMPA mean currents by
the antagonists was significant relative to control (P <
0.05, 0.01, and 0.001, respectively, by t-test). The
blocking effects of 38 or 1 were fully reversible within
20-30 min of antagonist washout (data not shown).
Neither agent tested alone showed any effect on steady
holding current at -80 mV.
ED50
TD50
TIc
compd
clonic phase
locomotor deficit
TD50/ED50
24b
25b
26b
27
28
29
30
32b
35
36
38
39
40
42
43
1
75.5 (47.5-120)
33.9 (26.0-44.2)
102 (76.1-137)
110 (79.3-151)
197 (138-281)
142 (87.3-231)
240 (102-564)
226 (116-437)
2.6
4.2
2.3
2.0
ND
ND
ND
4.1
ND
ND
>120
>150
>120
>120
>150
>150
Discu ssion
37.8 (23.7-60.1)
154 (104-227)
The recent availability of centrally active antagonists
of non-NMDA (AMPA/kainate) receptors has made it
possible to evaluate the potential of such compounds as
antiepileptic agents in animal seizure models. Several
previous studies4,6,17-19 have indicated that the systemic
administration of AMPA receptor antagonists to experi-
mental animals produces anticonvulsant activity; the
present results would confirm these effects.
Several 2,3-benzodiazepine derivatives reported in the
present study have marked anticonvulsant activity, in
some cases higher than compound 1, both in the
audiogenic seizure test with DBA/2 mice (Table 1) and
against the maximal electroshock- and pentylenetetra-
zole-induced seizures in Swiss mice (Table 2).
>120
>120
>150
>150
15.0 (9.01-24.0)
56.8 (39.3-82.1) 3.8
51.5 (34.1-77.8) 2.7
19.3 (16.9-22.0)
50.2 (34.6-73.0)
101 (52.0-194)
159 (95-268)
181 (106-309)
3.2
1.8
ND
2.1
>120
35.8 (24.4-52.4)
>150
76.1 (47.5-122)
a
All data are expressed as µmol/kg and were calculated
according to the method of Litchfield and Wilcoxon.41 Reference
b
1. c TI ) therapeutic index and represents the ratio between TD50
and ED50 (from the clonic phase of the audiogenic seizures); ND
) not determined.
receptors; [3H]-(RS)-3-(2-carboxypiperazin-4-yl)propane-
1-phosphonic acid ([3H]CPP) and [3H]dizocilpine ([3H]-
MK-801) from NMDA receptors; [3H]-5,7-dichloro-
kynurenic acid ([3H]5,7-DCKA) from glycine site on
NMDA receptors; [3H]AMPA and [3H]-6-cyano-7-nitro-
quinoxaline-2,3-dione ([3H]CNQX) from AMPA/kainate
receptors; mixture of [3H](1S,3R)ACPD and [3H](1R,-
3S)ACPD from metabotropic glutamate receptors.
Effects on Motor Movem en ts. Table 6 shows the
doses which induced motor toxicity in 50% of mice (TD50
values) obtained 30 min following the ip administration
of various 2,3-benzodiazepines. Compounds 24, 26-27,
39, 42, and 1 affected the rotarod test at doses ap-
proximately double those which showed anticonvulsant
activity while derivatives 25, 32, 38, and 40 had
therapeutic index (TI) values in the range 3.2-4.2.
Effects of 2,3-Ben zod ia zep in e Der iva tives on
AMP A Recep tor -Med ia ted Mem br a n e Cu r r en ts, in
Vitr o. The effects of 38, the most active compound of
the series, and 1 were compared on AMPA receptor-
mediated membrane currents measured under voltage
clamp in guinea pig olfactory cortical neurons in vitro.
Inward currents induced by bath application of AMPA
were recorded in the presence of 1 µM tetrodotoxin
(TTX) to prevent voltage-activated sodium currents and
repetitive sodium spikes at the peak of AMPA re-
sponses. When applied to neurons voltage clamped at
-80 mV holding potential, AMPA (0.25-5 µM; 4 min;
N ) 7 experiments) produced a slow dose-dependent
inward current of large amplitude (mean amplitude at
1 µM; 0.73 ( 0.12 nA) that attained a plateau over 3-4
min of application and declined slowly to baseline
current level over 5-10 min after drug washout (often
with a slow outward “rebound” current phase, Figure
4). This response was reproducible, providing an ad-
equate period (15-20 min) of intermediate washout was
allowed. In the presence of a fixed concentration (50
µM) of 38 (N ) 4 experiments; 15 min preincubation),
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. A simi-
The structure-activity relationships in this series
were examined by varying C-8, N-3, and C-1 aromatic
ring substitutions in order to test the influence of
physicochemical parameters on anticonvulsant activity.
One or two methoxy groups were introduced in the 7-
and 8-positions of the benzodiazepine moiety; H at N-3
was substituted with a methyl or an acetyl group, and
in particular, a number of substitutions at the C-4′
position of the phenyl ring at C-1 were explored.
Finally, since the 4′-aminophenyl derivative proved to
be the most active compound of the series, the position
of the NH2 substituent was also varied.
The ability of the structural changes introduced into
the 2,3-benzodiazepines to modify the anticonvulsant
efficacies of the parent compounds is shown in Tables
1 and 2 and discussed below.
The presence of two methoxy groups on the benzodi-
azepine system increases anticonvulsant activity; in
fact, the dimethoxy-substituted compound 25 is more
active than the monomethoxy derivative 24 and shows
an anticonvulsant activity comparable to that of 1.
The introduction of a bromine or a chlorine atom and
a cyano or a nitro group on the phenyl ring at C-1
negatively influences the activity, whereas the presence
of an amino group, independently by its position,
increases the anticonvulsant activity of the unsubsti-
tuted parent compound 25 (Tables 1 and 2), the most
active compounds being the amino derivatives 38 and
39, as with other amino derivatives.20 It is interesting
to note that 38 and 39 proved to be more potent than
compound 1.
The presence of a methyl and in particular an acetyl
group at N-3 generally led to compounds with weaker
anticonvulsant properties compared to the N-3 unsub-
stituted derivatives. The time course studied for de-
rivatives 40 and 44 suggests (Figure 1) that a metabolic
activation might take place in vivo and that the N-
substituted derivatives undergo biotransformation in
the parent compounds by loss of the N-3 substituent.
We have chemical evidence from an HPLC study that

1264 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Chimirri et al.
F igu r e 4. 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 38 (upper panel) or in absence (3; N
) 3) and presence (1; N ) 3) of 50 µM 1 (lower panel). Each point represents mean ((SEM) plotted against AMPA concentration
(0.25-5 µM; log scale). Points without error bars had 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 15 min preincubation with 50 µM compound 38 (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;
Students t-test). Scale bar applies to all traces. Drug washout periods were recorded at 0.4 × chart speed.
compounds 40 and 40 are converted to compound 38 in
rats (unpublished data), and this could better explain
the results of the behavioral time course effects. Un-
expectedly, the presence of a methyl group at the N-3
position in compounds 33 and 34 enhances the activity
with respect to the related unsubstituted 26 and 27
derivatives (Table 1). The reason for this different
behavior with respect to all the other N-substituted
derivatives is not presently understood. Further ex-
periments on these compounds and their metabolites
are in progress and will be detailed in future reports.
40, and 44, become more potent 30 min after pretreat-
ment and showed a more prolonged time course of action
than 1.
Data supporting the hypothesis that the 2,3-benzo-
diazepine derivatives tested interact with non-NMDA
receptors are also reported. Specifically, the drugs were
found to afford effective protection against seizures
induced by AMPA, a chemical agent which stimulates
the AMPA/kainate receptor complex (Table 3).
In the present study we also demonstrated that
aniracetam, a potentiator of AMPA effect,14 markedly
antagonized the anticonvulsant effects of 25, 32, 38, and
40 in DBA/2 mice (Table 4) with a pattern of activity
similar to 1 and NBQX7,18 and shifted to the right the
dose-response curves for derivatives 38 and 40 (Figure
3). Since 1 and NBQX are considered as AMPA/kainate
receptor antagonists,6,17,18,21 we suggest that 2,3-ben-
zodiazepines also act as antagonists at this receptor site.
It has been shown that aniracetam is a positive allos-
teric modulator of AMPA/kainate-selective glutamate
receptors by reducing the rate of rapid receptor au-
todensensitization14 and that it reverses the anticon-
vulsant properties of some 2,3-benzodiazepines (Table
4), we therefore suggest that, by analogy to 1,20,22-23 the
2,3-benzodiazepines described here might antagonize
The different anticonvulsant potencies of the 2,3-
benzodiazepines were only partially correlated to their
relative lipophilicities (Table 1), indicating intrinsic
differences in the anticonvulsant potencies of the com-
pounds, rather than merely a different degree of pen-
etration through the blood-brain barrier following sys-
temic administration.
Figure 1 shows that when anticonvulsant activity was
assessed 15 min after treatment with drug, compound
1 demonstrated an anticonvulsant potency higher than
other 2,3-benzodiazepines, indicating faster blood-brain
barrier transport, or higher affinities for the AMPA
receptor,6 whereas the 2,3-benzodiazepines tested, 38,

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1265
the AMPA/kainate receptor-mediated responses by an
allosteric blocking mechanism. Indeed, the noncompeti-
tive nature of the AMPA block exerted by the most
potent of the 2,3-benzodiazepine derivative tested (38)
was confirmed in electrophysiological experiments per-
formed in olfactory cortical brain slice neurons. In these
tests, both 38 and 1 consistently depressed the AMPA
dose-response relation in a nonparallel manner, even
at the highest agonist concentration used (5 µM). This
noncompetitive mode of antagonism of AMPA responses
by 1 detected here is in agreement with previous in vitro
data obtained from cultured hippocampal neurones,23,24
cortical wedges,25 and hippocampal slices.26 The simi-
larity between 38 and 1 effects further implies that the
2,3-benzodiazepine analogues may all share a common
noncompetitive mode of action at the AMPA receptor.
Electrophysiological experiments with the derivative 38
confirm this hypothesis.
In conclusion, the novel 2,3-benzodiazepine deriva-
tives reported in this study have marked anticonvulsant
activity both in the audiogenic seizures test with DBA/2
mice and against the maximal electroshock- and pen-
tylenetetrazole-induced seizures in Swiss mice. In
particular, derivatives 25 and 32 showed activities
comparable to that of 1, and compounds 38 and 39 were
more potent than 1 in vivo. They also showed longer
lasting anticonvulsant action and less toxicity than this
compound. Moreover, they showed significant anticon-
vulsant activity against seizures induced by AMPA, thus
suggesting an involvement of AMPA receptors, whereas
they had no significant effects on BZRs. Electrophysi-
ological data suggest a noncompetitive blocking mech-
anism of the 2,3-benzodiazepines at the AMPA receptor
site at the doses studied. The structural features which
determine the best biological profile in this class of
compounds seem to be as follows: two methoxy groups
at C-7 and C-8, an amino group on the phenyl ring at
C-1, and a hydrogen atom at N-3 position. Further
studies aimed at better elucidating the distribution,
metabolism, and mechanism of action of 2,3-benzodi-
azepines are currently in progress.
The anticonvulsant activity of these compounds was
evident at doses which generally did not cause sedation
and ataxia. It has long been known that antagonists
of the EAA receptors, especially those which block ion
channels [e.g. dizocilpine (MK-801)], can induce cogni-
tive deficits and a variety of other neurological and
behavioral side effects.22,27 There are, however, studies
showing that potent antagonists at the AMPA/kainate
receptor have anticonvulsant effects at doses below
those impairing behavior.28 The anticonvulsant profiles
and improved therapeutic indices of some of the new
2,3-benzodiazepines provide an interesting illustration
of the research effort which is being made to discover
new drugs interacting with EAA receptors and possess-
ing therapeutic potential with lower side effects.4,22,29
The therapeutic indices (TI) of the present compounds
were similar to that of 1 with the exception of 25, 32,
38, and 40, which caused significantly less motor
impairment in the rotarod test and had TI values
approximately twice that of 1 (Table 6). Since the
anticonvulsant effects of 25, 38-40, and 1 were not
reversed by flumazenil under an identical dose regimen
(Table 5), it seems likely that the actions of these
derivatives were not mediated by benzodiazepine recep-
tor sites. The latter consideration derives also from the
following observations: (a) the amounts of 2,3-benzo-
diazepines used in this study had not significant effects
on the GABA/benzodiazepine receptor complex; (b) the
2,3-benzodiazepines showed significant anticonvulsant
activity against AMPA; (c) the anticonvulsant effects
were reversed by aniracetam but not by flumazenil.
Since AMPA/kainate receptor antagonists have been
claimed to be more effective than NMDA receptor
antagonists in reducing damage in models of global
ischaemia,30 the present data would support the sug-
gestion that the neuroprotective effects of 2,3-benzodi-
azepines are mediated through the AMPA/kainate
receptor complex.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Kofler
hot stage apparatus and are uncorrected. Elemental analyses
were made on a Carlo Erba 1106 elemental analyzer for C, H,
and N, and the results are within (0.4% of the theoretical
values. 1H NMR spectra were recorded in CDCl3 on a Varian
Gemini-300 spectrometer. Chemical shifts were expressed in
δ (ppm) relative to TMS as internal standard. All exchange-
able protons were confirmed by addition of D2O.
Compounds 10-12, 17-19, 24-26, and 31-33 were pre-
pared according to a procedure previously described.15
Gen er a l P r oced u r e for th e Syn th esis of 2-Ar ylisoch -
r om a n s 13-16. A solution of 3,4-dimethoxyphenethyl alcohol
3 (1.82 g 10 mmol) and the suitable aldehyde 6-9 (12 mmol)
in anhydrous dioxane (30 mL) was first saturated with gaseous
HCl and then refluxed for 1 h. After cooling, the reaction
mixture was poured into water (100 mL), made alkaline with
2 N NaOH (20 mL), and extracted with EtOAc (3 × 60 mL).
The organic phase was dried over MgSO4, the solvent was
removed under reduced pressure, and the resulting residue
was crystallized from EtOH to provide compounds 13-16.
1-(4′-Br om op h en yl)-6,7-d im et h oxyisoch r om a n (13).
Mp: 109-111 °C. Yield: 44%. 1H NMR: 2.73 and 3.00 (2m,
2H, CH2-4), 3.67 and 3.88 (2s, 6H, OCH3-7 and OCH3-8), 3.89
and 4.10 (2m, 2H, CH2-3), 5.64 (s, 1H, H-1), 6.18 (s, 1H, H-6),
6.65 (s, 1H, H-9), 7.17-7.50 (m, 4H, Ar). Anal. (C17H17BrO3)
C, H.
1-(4′-Cya n op h en yl)-6,7-d im et h oxyisoch r om a n
(14).
Mp: 116-118 °C. Yield: 92%. 1H NMR: 2.73 and 3.04 (2m,
2H, CH2-4), 3.66 and 3.88 (2s, 6H, OCH3-7 and OCH3-8), 3.89
and 4.09 (2m, 2H, CH2-3), 5.71 (s, 1H, H-1), 6.14 (s, 1H, H-6),
6.67 (s, 1H, H-9), 7.42-7.67 (m, 4H, Ar). Anal. (C18H17NO3)
C, H, N.
1-(4′-Nit r op h en yl)-6,7-d im et h oxyisoch r om a n
(15).
Despite the lack of activity of the 2,3-benzodiazepines
at dopamine, serotonin, noradrenaline, NMDA, glycine
site on NMDA and metabotropic glutamate binding
sites, an interaction at other sites involved in the
generation or expression of seizures cannot presently
be ruled out. Since no reliable ligand-binding assay has
been reported for noncompetitive agonists and antago-
nists affecting the AMPA receptor,13 our binding data
do not exclude that these new series of 2,3-benzodiaz-
epines may act as noncompetitive AMPA antagonists.
Mp: 138-140 °C. Yield: 77%. 1H NMR: 2.76 and 3.07 (2m,
2H, CH2-4), 3.66 and 3.88 (2s, 6H, OCH3-7 and OCH3-8), 3.89
and 4.12 (2m, 2H, CH2-3), 5.76 (s, 1H, H-1), 6.14 (s, 1H, H-6),
6.68 (s, 1H, H-9), 7.48-8.23 (m, 4H, Ar). Anal. (C17H17NO5)
C, H, N.
1-(3′-Nit r op h en yl)-6,7-d im et h oxyisoch r om a n
(16).
Mp: 110-111 °C. Yield: 85%. 1H NMR: 2.76 and 3.07 (2m,
2H, CH2-4), 3.66 and 3.89 (2s, 6H, OCH3-7 and OCH3-8), 3.67
and 4.13 (2m, 2H, CH2-3), 5.78 (s, 1H, H-1), 6.16 (s, 1H, H-6),
6.68 (s, 1H, H-9), 7.50-8.20 (m, 4H, Ar). Anal. (C17H17NO5)
C, H, N.

1266 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Chimirri et al.
Gen er a l P r oced u r e for th e Syn th esis of 2-Ar oylp h e-
n yla cetic Acid s 20-23. To a cooled stirred solution of 13-
16 (0.002 mol) in acetone was added dropwise under 5 °C a
solution of CrO3 in 35% H2SO4. After the addition, the mixture
was stirred for 2 h. The reaction mixture was poured into ice
water. The resulting precipitate was filtered, washed with
water, and crystallized from benzene to give 20-23.
2-(4′-Br om op h en yl)-4,5-d im et h oxyp h en yla cet ic Acid
(20). Mp: 228-230 °C. Yield: 72%. 1H NMR: 3.76 (s, 2H,
CH2), 3.78 and 3.97 (2s, 6H, OCH3-7 and OCH3-8), 6.89 (s, 1H,
3.97 (2s, 6H, OCH3-7 and OCH3-8), 6.59 (s, 1H, H-6), 6.89 (s,
1H, H-9), 7.60-8.54 (m, 4H, Ar). Anal. (C18H17N3O5) C, H,
N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
38-41. To a mixture of the appropriate nitro derivative 29,
30, 36, or 37 (0.002 mol) and granulated tin (0.004 mol) was
dropwise added 37% HCl (3 mL). The reaction mixture was
heated on a boiling water bath for 1 h. The mixture was
cooled, treated with NaOH, and extracted with CHCl3. The
organic phase was dried over Na2SO4, the solvent was evapo-
rated, and the crude residue was purified by column chroma-
tography (EtOAc/CCl4, 70:30, as eluent).
H-6), 6.96 (s, 1H, H-9), 7.63-7.71 (m, 4H, Ar). Anal. (C17H15
BrO5) 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 -4-on e (38). Mp: 178-180 °C. Yield: 59%.
1H NMR: 3.47 (s, 2H, CH2), 3.74 and 3.96 (2s, 6H, OCH3-7
and OCH3-8), 3.97 (br s, 2H, NH2), 6.69-7.46 (m, 6H, Ar), 8.32
(br s, 1H, NH). Anal. (C17H17N3O3) C, H, N.
2-(4′-Cya n op h en yl)-4,5-d im et h oxyp h en yla cet ic a cid
(21). Mp: 153-155 °C. Yield: 24%. 1H NMR: 3.78 and 3.99
(2s, 6H, OCH3-7 and OCH3-8), 3.81 (s, 2H, CH2), 6.86 (s, 1H,
H-6), 6.99 (s, 1H, H-9), 7.80-7.94 (m, 4H, Ar). Anal. (C18H15
-
NO5) 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 -4-on e (39). Mp: 253-255 °C. Yield: 56%.
1H NMR: 3.49 (s, 2H, CH2), 3.74 and 3.96 (2s, 6H, OCH3-7
and OCH3-8), 3.82 (br s, 2H, NH2), 6.71-7.22 (m, 6H, Ar), 8.58
(br s, 1H, NH). Anal. (C17H17N3O3) C, H, N.
2-(4′-Nitr oph en yl)-4,5-dim eth oxyph en ylacetic acid (22).
Mp: 170-172 °C. Yield: 25%. 1H NMR: 3.77 and 3.99 (2s,
6H, OCH3-7 and OCH3-8), 3.86 (s, 2H, CH2), 6.86 (s, 1H, H-6),
6.95 (s, 1H, H-9), 7.96-8.36 (m, 4H, Ar). Anal. (C17H15NO7)
C, H, N.
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-b en zod ia zep in -4-on e (40). Mp: 223-225 °C.
Yield: 78%. 1H NMR: 3.39 (s, 3H, NCH3) 3.47 (br s, 2H, CH2),
3.75 and 3.95 (2s, 6H, OCH3-7 and OCH3-8), 3.93 (br s, 2H,
NH2), 6.68-7.49 (m, 6H, Ar). Anal. (C18H19N3O3) C, H, N.
1-(3′-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-b en zod ia zep in -4-on e (41). Mp: 143-145 °C.
Yield: 80%. 1H NMR: 3.43 (s, 3H, NCH3) 3.52 (br s, 2H, CH2),
3.75 and 3.96 (2s, 6H, OCH3-7 and OCH3-8), 6.71-7.23 (m,
6H, Ar). Anal. (C18H19N3O3) C, H, N.
Gen er a l P r oced u r e for t h e Syn t h esis of 3-Acet yl
Der iva tives 42-44. To a cooled stirred solution of compound
25, 28, or 38 (0.001 mol) in CHCl3 (30 mL) and Et3N (5 mL)
was dropwise added Ac2O (5 mL). The mixture reaction was
stirred at room temperature for 1-2 h, poured into water,
extracted with CHCl3, and dried over Na2SO4. Evaporation
of the solvent under reduced pressure afforded a crude product,
which was purified by treatment with Et2O.
2-(3′-Nitr oph en yl)-4,5-dim eth oxyph en ylacetic Acid (23).
Mp: 203-205 °C. Yield: 54%. 1H NMR: 3.78 and 3.99 (2s,
6H, OCH3-7 and OCH3-8), 3.84 (s, 2H, CH2), 6.89 (s, 1H, H-6),
6.97 (s, 1H, H-9), 7.70-8.64 (m, 4H, Ar). Anal. (C17H15NO7)
C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 2,3-Ben zod i-
a zep in -4-on es 27-30 a n d 34-37. The appropriate 2-aroyl-
phenylacetic acid (0.003 mol) was refluxed with hydrazine
hydrate or monomethylhydrazine (0.008 mol) for 2-3 h in
EtOH (50 mL). After cooling, the solvent was removed under
reduced pressure and the residue crystallized from MeOH.
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 -4-on e (27). Mp: 221-223 °C. Yield: 75%.
1H NMR: 3.50 (s, 2H, CH2), 3.73 and 3.96 (2s, 6H, OCH3-7
and OCH3-8), 6.62 (s, 1H, H-6), 6.84 (s, 1H, H-9), 7.50-7.58
(m, 4H, Ar), 8.43 (br s, 1H, NH). Anal. (C17H15BrN2O3) C, H,
N.
1-(4′-Cya n 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 -4-on e (28). Mp: 245-247 °C. Yield: 38%.
1H NMR: 3.51 (s, 2H, CH2), 3.73 and 3.97 (2s, 6H, OCH3-7
and OCH3-8), 6.56 (s, 1H, H-6), 6.85 (s, 1H, H-9), 7.71-7.79
(m, 4H, Ar), 8.63 (br s, 1H, NH). Anal. (C18H15N3O3) 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 -4-on e (29). Mp: 250-252 °C. Yield: 77%.
1H NMR: 3.53 (s, 2H, CH2), 3.72 and 3.98 (2s, 6H, OCH3-7
and OCH3-8), 6.56 (s, 1H, H-6), 6.86 (s, 1H, H-9), 7.82-8.30
(m, 4H, Ar), 8.60 (br s, 1H, NH). Anal. (C17H15N3O5) 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 -4-on e (30). Mp: 263-265 °C. Yield: 88%.
1H NMR: 3.53 (s, 2H, CH2), 3.72 and 3.98 (2s, 6H, OCH3-7
and OCH3-8), 6.59 (s, 1H, H-6), 6.88 (s, 1H, H-9), 7.60-8.53
(m, 4H, Ar), 8.67 (br s, 1H, NH). Anal. (C17H15N3O5) C, H, N.
1-(4′-Br om op h en yl)-3,5-d ih yd r o-7,8-d im eth oxy-3-m eth -
yl-4H -2,3-b en zod ia zep in -4-on e (34). Mp: 154-156 °C.
Yield: 79%. 1H NMR: 3.43 (s, 5H, N-CH3 and CH2), 3.74 and
3.96 (2s, 6H, OCH3-7 and OCH3-8), 6.62 (s, 1H, H-6), 6.86 (s,
1H, H-9), 7.51-7.59 (m, 4H, Ar). Anal. (C18H17BrN2O3) C, H,
N.
1-(4′-Cya n op h en yl)-3,5-d ih yd r o-7,8-d im eth oxy-3-m eth -
yl-4H -2,3-b en zod ia zep in -4-on e (35). Mp: 206-208 °C.
Yield: 45%. 1H NMR: 3.46 (s, 5H, NCH3 and CH2), 3.73 and
3.96 (2s, 6H, OCH3-7 and OCH3-8), 6.57 (s, 1H, H-6), 6.87 (s,
1H, H-9), 7.71-7.80 (m, 4H, Ar). Anal. (C19H17N3O3) C, H,
N.
3,5-Dih ydr o-7,8-dim eth oxy-3-m eth yl-1-(4′-n itr oph en yl)-
4H -2,3-b en zod ia zep in -4-on e (36). Mp: 230-232 °C.
Yield: 69%. 1H NMR: 3.48 (s, 5H, N-CH3 and CH2), 3.73 and
3.97 (2s, 6H, OCH3-7 and OCH3-8), 6.57 (s, 1H, H-6), 6.88 (s,
1H, H-9), 7.84-8.30 (m, 4H, Ar). Anal. (C18H17N3O5) C, H,
N.
3-Acet yl-3,5-d ih yd r o-7,8-d im et h oxy-1-p h en yl-4H -2,3-
ben zod ia zep in -4-on e (42). Mp: 154-156 °C. Yield: 65%.
1H NMR: 2.58 (s, 3H, COCH3), 3.59 (s, 2H, CH2), 3.75 and
3.98 (2s, 6H, OCH3-7 and O CH3-8), 6.69 (s, 1H, H-6), 6.90 (s,
1H, H-6), 7.42-7.73 (m, 4H, Ar). Anal. (C19H18N2O4) C, H,
N.
3-Acetyl-1-(4′-cyan oph en yl)-3,5-dih ydr o-7,8-dim eth oxy-
4H-2,3-ben zod ia zep in -4-on e (43). Mp: 98-100 °C. Yield:
72%. 1H NMR: 2.59 (s, 3H, COCH3), 3.59 (s, 2H, CH2), 3.75
and 3.99 (2s, 6H, OCH3-7 and OCH3-8), 6.60 (s, 1H, H-6), 6.92
(s, 1H, H-9), 7.73-7.87 (m, 4H, Ar). Anal. (C20H17N3O4) C,
H, N.
3-Acetyl-1-(4′-(d ia cetyla m in o)p h en yl)-3,5-d ih yd r o-7,8-
d im eth oxy-4H-2,3-ben zod ia zep in -4-on e (44). Mp: 148-
150 °C. Yield: 68%. 1H NMR: 2.22 (2s, 6H, NCOCH3) 2.57
(s, 3H, COCH3), 3.58 (s, 2H, CH2), 3.76 and 3.98 (2s, 6H,
OCH3-7 and OCH3-8), 6.70 (s, 1H, H-6), 6.89 (s, 1H, H-9), 7.63-
7.71 (m, 4H, Ar). Anal. (C23H23N3O6) 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.31 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
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.32 DBA/2 mice (8-12 g; 22-25-days-old) were pur-
3,5-Dih ydr o-7,8-dim eth oxy-3-m eth yl-1-(3′-n itr oph en yl)-
4H -2,3-b en zod ia zep in -4-on e (37). Mp: 263-265 °C.
Yield: 65%. 1H NMR: 3.48 (s, 5H, N-CH3 and CH2), 3.72 and

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8 1267
chased 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 drugs. 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 60 s was allowed for habituation and assessment of
locomotor activity. 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 general-
ized myoclonus and tonic flexion and extension sometimes
followed by respiratory arrest. The control and drug-treated
mice were scored for latency to and incidence of the different
phases of the seizures.33 The time course of the anticonvulsant
action of 38, 40, 44, and 1 was determined following the
administration of 33 µmol/kg of 2,3-benzodiazepines to groups
of 10 mice that were tested for sound-induced seizure re-
sponses at 5-180 min after drug administration.
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.34 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 eight to 10 mice
for each dose.
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 2,3-benzodiazepines 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 pentylenetetrazole (85 mg/kg) was applied
and the animals 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 pentylenetet-
razole seizure threshold.35
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:36 injections of drugs by this procedure
led to an 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.
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 2,3-benzodiazepines 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 2,3-benzodiazepines 25 and 38-40 bind to
receptors other than BZR was studied in crude rate brain
synaptic membranes according to established protocols37 by
using [3H]spiperone, [3H]ketanserin, [125I]pindolol, [3H]CPP,
[3H]MK-801, and [3H]AMPA. In other binding studies aliquots
of membrane suspension were 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). Nonspe-
cific 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.
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
inability of the mice to remain on the rotarod for a 2 min test
period.38 The ability of the mice to remain on the rotarod was
tested 30 min after administration of various 2,3-benzodiaz-
epines.
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 described39 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,40
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, 38,
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 38 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.
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 incubation medium containing 1 nmol
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 tem-
peratures). The ED50 values of each phase of the audiogenic
seizure or seizures induced by electroshock or pentylenetet-
razole was determined for each dose of compound adminis-
tered, and dose-response curves were fitted using a computer
program by the method of Litchfield and Wilcoxon.41 The

1268 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 8
Chimirri et al.
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data of electrophysiology are expressed as mean ( standard
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