Synthesis and anticonvulsant activity of novel and potent 2,3- benzodiazepine AMPA/kainate receptor antagonists

Article abstract of DOI:10.1021/jm991086d

We have previously shown that 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H- 2,3-benzodiazepin-4-ones (3) possess marked anticonvulsant properties and antagonize seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4- yl)propionic acid (AMPA) in analogy t

Full text of DOI:10.1021/jm991086d

4414
J . Med. Chem. 1999, 42, 4414-4421
Syn th esis a n d An ticon vu lsa n t Activity of Novel a n d P oten t 2,3-Ben zod ia zep in e
AMP A/Ka in a te Recep tor An ta gon ists
Silvana Grasso,*,∇ Giovambattista De Sarro,§ Angela De Sarro,† Nicola Micale,∇ Maria Zappala`,∇ Giulia Puia,‡
Mario Baraldi,‡ and Carlo De Micheli
Dipartimento Farmaco-Chimico and Istituto di Farmacologia, Universita` di Messina, Italy, Dipartimento di Medicina
Sperimentale e Clinica, Universita` di Catanzaro, Italy, Dipartimento di Scienze Farmaceutiche, Universita` di Modena, Italy,
and Istituto di Chimica Farmaceutica, Universita` di Milano, Italy
Received May 27, 1999
We have previously shown that 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-
4-ones (3) possess marked anticonvulsant properties and antagonize seizures induced by
2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) in analogy to the structurally
related 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466),
a well-known noncompetitive AMPA/kainate receptor antagonist. We now report the synthesis
of 3-(N-alkylcarbamoyl)-1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones
(4a -h ) and 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepine-4-thiones (5a -c). The
activity of all compounds, intraperitoneally (ip) injected, was evaluated against audiogenic
seizures in DBA/2 mice and against seizures induced by maximal electroshock (MES) and
pentylenetetrazole (PTZ) in Swiss mice. Some of the new compounds 4 and 5 showed remarkable
anticonvulsant activity, and their toxicity, as evidenced by the rotarod test, is lower than that
of 1. The time course of anticonvulsant activity of derivatives 4b and 5b,c was studied and
compared to that of 1 and 3b,c. Compounds 4a ,b and 5a -c antagonize seizures induced by
AMPA and kainate (KA) and their anticonvulsant activity is reversed by pretreatment with
aniracetam. Using the patch-clamp technique, the capability of derivatives 3c, 4b, and 5c to
antagonize KA-evoked currents in primary cultures of granule neurons was tested and compared
with that of the parent compounds 1 and 1-(4-aminophenyl)-3,4-dihydro-4-methyl-3-methyl-
carbamoyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (2, GYKI 53655).
Ch a r t 1
In tr od u ction
Overstimulation of ionotropic glutamate receptors,
N-methyl-D-aspartic acid (NMDA), 2-amino-3-(3-hy-
droxy-5-methylisoxazol-4-yl)propionic acid (AMPA), and
kainate (KA) receptors, is believed to be implicated in
the development of several neurodegenerative disorders
including epilepsy, stroke, and Alzheimer’s disease.1
1-(4-Aminophenyl)-4-methyl-7,8-methylenedioxy-5H-
2,3-benzodiazepine (1, GYKI 52466) (Chart 1), a 2,3-
benzodiazepine derivative, is the prototype of selective
noncompetitive AMPA receptor antagonists acting via
an allosteric site on the receptor complex.2 It possesses
remarkable anticonvulsant properties3 and behaves as
a neuroprotective agent in focal and global ischemia.4
A significant improvement in the pharmacological pro-
file of GYKI 52466 was obtained by appending an
alkylcarbamoyl group at N-3 of the benzodiazepine
ring.5 As a matter of fact, the 3-N-methylcarbamoyl
derivative 2 (GYKI 53655, LY 300168) (Chart 1) emerged
as the most potent compound among this series of
derivatives.6 Its ED50 value against KA-induced seizures
and in the maximal electroshock seizure test is 2-3-
fold higher than that of 1. As a blocker of the AMPA
and KA currents, 2 is 5-8-fold more potent than its
parent compound 1.
As part of a program aimed at identifying potent and
selective AMPA receptor antagonists,7,8 we have re-
cently reported9 an investigation on a series of 1-aryl-
3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-
4-ones (3) where the iminohydrazone portion of 1 was
replaced by an iminohydrazide moiety. In this context
* To whom correspondence should be addressed: S. Grasso, Dipar-
timento Farmaco-Chimico, Universita` di Messina,Viale Annunziata,
98168 Messina, Italy. Phone: +39 090 6766470. Fax: +39 090 355613.
E-mail: grasso@pharma.unime.it.
∇ Dipartimento Farmaco-Chimico.
§ Dipartimento di Medicina Sperimentale e Clinica.
† Istituto di Farmacologia.
‡ Dipartimento di Scienze Farmaceutiche.
Istituto di Chimica Farmaceutica.
10.1021/jm991086d CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/23/1999

Benzodiazepine AMPA/ Kainate Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4415
Sch em e 1a
(PTZ) in Swiss mice. We also assessed the propensity
of all compounds to induce neurological impairment by
using the rotarod test. The time course of anticonvulsant
activity of 3b,c, 4b, and 5b,c was studied and compared
to that of reference compound 1. The activity of 4a ,b
and 5a -c was also evaluated through their ability to
antagonize seizures induced by the intracerebroven-
tricular (icv) administration of AMPA or subcutaneous
(sc) administration of KA. In addition, the ability of
aniracetam (Ro 13-5057), a potentiator of the AMPA
effects,12 to reverse the anticonvulsant properties of
these compounds was studied.
To investigate at the cellular level the mechanism of
action of the in vivo active substances, we have used
the patch-clamp technique in primary cultures of gran-
ule neurons. The capability of 3c, 4b, and 5c to
antagonize KA-evoked current was tested and compared
with that of parent compounds 1 and 2.
Ch em istr y
2,3-Benzodiazepines 3d ,e, used as starting material,
were prepared via Friedel-Crafts acylation of methyl
3,4-methylenedioxyphenylacetate with 3- or 4-nitroben-
zoic acid, following a methodology previously reported,13
and subsequently reacted with an excess of hydrazine.
These intermediates were then treated with an excess
of the appropriate alkyl isocyanate in the presence of
triethylamine to yield the corresponding N-3-alkylcar-
bamoyl derivatives, which were then transformed into
final derivatives 4a -h (Scheme 1) by a catalytic reduc-
tion of their nitro group. Compounds 3a -c were pre-
pared as previously reported9 and converted into the
corresponding thiocarbonyl derivatives 5a -c by treat-
ment with Lawesson’s reagent at reflux in toluene
(Scheme 1). The elemental analysis (C, H, N) and 1H
NMR spectral data of synthesized compounds are in full
agreement with the proposed structures and are re-
ported in the Experimental Section.
a
(a) R3NCO/Et3N, CH2Cl2, rt, 36 h; (b) H2/5% Pd-C, MeOH,
rt, 3 h; (c) Lawesson’s reagent, toluene, reflux, 2 h.
we noticed that 3-aminophenyl and 4-aminophenyl
derivatives 3b,c are roughly 2-fold more potent than
both 1 and parent compound 3a . As deduced from the
rotarod test, compounds 3 are all characterized by a
toxicity lower than that of 1.
Resu lts a n d Discu ssion
Novel 3-(N-alkylcarbamoyl)-1-aryl-3,5-dihydro-7,8-
methylenedioxy-4H-2,3-benzodiazepin-4-ones (4a-h ) and
1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodi-
azepine-4-thiones (5a -c) were tested for anticonvulsant
activity against audiogenic seizures induced in DBA/2
mice, and the results are compared with those previ-
ously reported9 for model compounds 1 and 3a -c (Table
1). The compounds were administered intraperitoneally
(ip) at various doses in the range of 3.3-200 µmol/kg
and their anticonvulsant properties, expressed as ED50,
evaluated 30 min after injection. A number of the new
derivatives are provided with remarkable anticonvul-
sant activity which is higher than that displayed by 1.
A survey of the results reported in Table 1 reveals that
the same modification carried out on derivatives 3b,c
produces an increase in the anticonvulsant activity only
in the case of methyl derivatives 4a ,b. A lengthening
of the side chain brought about a decrease in activity
in all the derivatives but 4g in which activity remained
unchanged. On the other hand, replacement of the
carbonyl group of 3a -c with a thiocarbonyl moiety was
always productive causing an increase in potency. This
result matches that previously reported for related
compounds.10 A conceivable explanation could be envis-
In analogy to what has been previously observed5 in
the series of GYKI 52466 analogues, where the intro-
duction of an alkylcarbamoyl group appended at the N-3
position of the heterocyclic nucleus brought about an
increase in potency, we prepared a series of 3-(N-
alkylcarbamoyl)-1-aryl-3,5-dihydro-7,8-methylenedioxy-
4H-2,3-benzodiazepin-4-ones (4a -h ) (Scheme 1). Fur-
thermore, since it has been reported10 that the bioisosteric
replacement of oxygen by sulfur in the carbonyl group
of derivatives structurally related to 3 gave a significant
improvement in the pharmacological profile, we syn-
thesized some 1-aryl-3,5-dihydro-7,8-methylenedioxy-
4H-2,3-benzodiazepine-4-thiones (5a -c). The new com-
pounds (4a -h and 5a -c) were evaluated for their
anticonvulsant properties 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 anticonvul-
sant drugs.11 Derivatives 4a -h and 5a -c were also
examined as antagonists against seizures induced either
by maximal electroshock (MES) or by pentylenetetrazole

4416 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grasso et al.
Ta ble 1. Anticonvulsant Activity of Compounds 1 and 3-5 Against Audiogenic Seizures in DBA/2 Mice, TD50 Values on Locomotion
Assessed by Rotarod Test, and Relative Lipophilicity (Rm)
ED50, µmol/kga
TD50, µmol/kg,a
locomotor deficit
TI,b
TD50/ED50
compd
clonic phase
tonic phase
Rm
1
35.8 (24.4-52.4)
43.3 (34.4-54.6)
18.0 (10.0-32.5)
15.4 (10.1-23.5)
13.6 (6.22-29.9)
12.4 (6.44-23.8)
44.9 (26.6-75.8)
35.0 (18.5-66.3)
85.3 (69.1-105)
69.4 (36.2-133)
18.1 (10.4-31.5)
38.7 (21.2-70.8)
23.8 (13.8-41.2)
16.7 (10.2-27.3)
11.8 (6.14-22.5)
25.3 (16.0-40.0)
40.6 (30.1-54.9)
12.7 (6.13-26.2)
10.9 (4.60-24.6)
11.2 (5.31-23.5)
8.70 (4.61-16.4)
32.1 (17.7-58.3)
23.8 (13.4-42.1)
63.8 (45.1-90.2)
49.2 (25.9-93.4)
12.7 (9.31-17.2)
32.6 (18.2-58.4)
18.8 (10.2-34.5)
8.88 (5.71-13.8)
5.09 (2.14-12.1)
76.1 (47.5-122)
159 (82.6-306)
101 (52.0-194)
99.1 (72.4-135)
48.9 (34.7-68.7)
48.6 (31.4-54.6)
126 (80.5-196)
134 (70.7-255)
>100
182 (95.1-350)
56.2 (39.4-80.0)
108 (82.2-143)
97.1 (73.2-129)
53.7 (32.9-87.6)
39.8 (25.0-63.3)
2.1
3.7
5.6
4.5
3.6
3.9
2.8
3.8
ND
2.6
3.1
2.8
4.1
3.2
3.4
-0.404
-0.215
-0.484
-0.552
-0.441
-0.459
-0.323
-0.368
-0.181
-0.250
-0.075
-0.110
-0.185
-0.213
-0.269
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
a
All data were calculated according to the method of Litchfield and Wilcoxon;27 95% confidence limits are given in parentheses. At
b
least 32 animals were used to calculate each ED50 and TD50 value. TI, therapeutic index, represents the ratio between TD50 and ED50
(from the clonic phase of the audiogenic seizures); ND, not determined.
Ta ble 2. Anticonvulsant Activity of Compounds 1 and 3-5
observed in the 4-aminophenyl-substituted derivatives
3c and 5c which showed their peak effect at 15 min and
returned to control seizure after 90-120 min from
administration. Noteworthy, the N-3-methylcarbamoyl
derivative 4b showed its maximum protection between
15 and 90 min with a return to control after 120 min
from administration. On the contrary the 3-aminophe-
nyl-substituted derivatives 3b and 5b displayed their
maximum protection in the range 30-45 min, and their
effect disappeared after 90-120 min. As a consequence,
if the comparison is referred to the data obtained after
15 min from administration, only derivatives 3c and 5c
are more active than reference compound 1, whereas if
we compare the results obtained in the range 30-60
min, all five compounds are more active than 1 (e.g.
ED50 ) 11.8 µmol/kg for 5c versus ED50 ) 35.8 µmol/kg
for 1).
To correlate the anticonvulsant activity of novel
compounds 4 and 5 with their affinity for AMPA
receptors, an additional test, based on AMPA-induced
seizures in DBA/2 mice, was performed (Table 4). As
shown in Figures 1 and 2, the clonic and tonic phases
of the seizures induced by icv administration of AMPA
were significantly reduced 30 min after ip administra-
tion of 4a ,b and 5a -c in analogy to 1.
Against MES- and PTZ-Induced Seizures in Swiss Mice
ED50, µmol/kga ((95% confidence limits)
compd
MES
PTZ
1
35.7 (29.3-43.4)
42.6 (26.4-68.8)
19.3 (16.9-22.0)
32.1 (23.2-44.3)
21.4 (12.6-36.3)
18.6 (8.40-41.2)
60.2 (19.5-185)
52.5 (40.4-68.2)
>100
76.5 (52.2-112)
26.6 (17.4-40.6)
79.7 (36.5-174)
37.8 (23.7-60.1)
30.9 (19.4-48.4)
9.79 (8.09-15.7)
68.3 (56.2-83.1)
78.0 (46.0-132)
40.5 (22.9-71.7)
71.8 (53.2-96.9)
19.6 (7.70-49.6)
16.3 (7.30-36.0)
90.7 (72.2-114)
68.2 (40.0-116)
>100
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
>100
36.4 (31.3-42.3)
>100
80.3 (47.9-134.6)
40.2 (24.4-66.2)
25.2 (15.2-43.5)
a
All data were calculated according to the method of Litchfield
and Wilcoxon.27 At least 32 animals were used to calculate each
ED50 value.
aged in an easier ip absorption and a more favorable
diffusion across the blood-brain barrier. As a matter
of fact, compounds 5a -c are provided with a higher
lipophilicity (expressed as Rm value) than parent de-
rivatives 3a -c (e.g. Rm ) -0.269 for 5c versus Rm
)
We also tested the influence of aniracetam, a poten-
tiator of the AMPA effect,12 on the anticonvulsant
activity of derivatives 4a ,b and 5a -c in DBA/2 mice
(Table 4). An icv injection of aniracetam (50 nmol/
mouse) on its own had no convulsant activity; neverthe-
less, the administration of aniracetam 60 min before the
injection of the tested compounds reversed their anti-
convulsant effects and shifted the dose-response curves
to the right with a pattern of activity similar to that of
1 and 3.
Furthermore, compounds 4a ,b and 5a -c produced a
dose-dependent protection against KA-induced seizures
(Table 4). The ED50 values are higher than those needed
to block audiogenic seizures (Table 1) and lower than
or similar to those capable of protecting the animals
against hind limb extension in the MES test (Table 2).
In addition, although few mice exhibited a lowering in
body temperature, a decrease in motor activity, and
unsteady gait after administration of the highest doses,
-0.552 for 3c; Table 1). However, lipophilicity is not the
only parameter able to affect the potency of the com-
pounds since in the series of N-3-carbamoyl derivatives
there is no correlation between lipophilicity and activity
(Table 1).
All compounds were also tested against MES- and
PTZ-induced seizures in Swiss mice. As shown in Table
2, the tonic extension and the clonic phase of the
seizures induced by MES and PTZ, respectively, were
significantly reduced 45 min after ip administration of
the tested compounds. It is noteworthy that compounds
4b and 5c, the most active of the series, show in both
tests activity 2-4-fold higher than that of the parent
compound 3c.
The time course of the anticonvulsant activity of 3b,c,
4b, and 5b,c was also studied and compared to that of
1 (Table 3). Model compound 1 displayed its maximum
protection at 15 min from ip administration and re-
turned to control at 90 min. The same trend was

Benzodiazepine AMPA/ Kainate Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4417
Ta ble 3. ED50 Values at Various Times following ip Administration of Compounds 1, 3b,c, 4b, and 5b,c
ED50, µmol/kg ((95% confidence limits),a clonic phase
compd
15 min
30 min
45 min
60 min
90 min
>50
47.1
(33.5-66.3)
>50
120 min
>50
1
10.8
(7.11-16.4)
>50
35.8
(24.4-62.4)
18.0*
(10.0-32.5)
15.4**
(10.1-23.5)
12.4**
(6.44-23.8)
16.7**
37.3
(27.2-52.1)
19.1*
(12.4-29.4)
18.7**
(11.3-31.0)
12.1**
(7.28-20.2)
15.2**
39.5
(29.6-52.7)
26.3
(21.1-32.9)
21.3*
(14.2-31.9)
13.0**
(7.40-22.8)
22.4*
3b
3c
4b
5b
5c
>50
>50
7.55
(4.08-14.0)
14.3
(7.54-27.0)
32.6
(25.2-42.1)
5.90*
19.4**
(7.96-47.5)
45.2
(31.4-65.1)
45.0
(34.1-59.4)
40.8
(13.2-126)
>50
(10.2-27.3)
11.8**
(6.14-22.5)
(11.5-20.1)
13.4**
(11.3-15.9)
(16.2-31.0)
19.6*
(12.8-30.1)
>50
(4.70-7.41)
a
Significant differences among compounds 1 and 3-5 were evaluated at the corresponding times and denoted as *p < 0.05 and **p <
0.001 using the method of Litchfield and Wilcoxon.27
Ta ble 4. ED50 Values of 1 and 3-5 Against AMPA- and KA-Induced Seizures and Against Audiogenic Seizures after Pretreatment
with Aniracetam in DBA/2 Mice
ED50, µmol/kga ((95% confidence limits)
AMPAb
pretreatment with aniracetamd
compd
clonic phase
tonic phase
KAc
clonic phase
tonic phase
1
57.5 (43.5-76.0)
70.3 (58.7-84.1)
29.2 (16.9-50.4)
37.9 (27.3-52.8)
23.4 (14.3-38.4)
18.6 (6.44-53.6)
46.3 (32.9-65.2)
32.8 (26.5-40.6)
15.6 (9.86-24.8)
40.5 (26.3-60.8)
56.9 (40.9-79.2)
23.8 (14.2-39.9)
28.5 (20.6-39.4)
14.2 (7.08-28.4)
10.3 (5.27-20.1)
35.4 (23.1-54.0)
26.2 (19.3-34.2)
11.5 (9.76-13.6)
27.8 (18.8-40.9)
49.9 (29.6-84.0)
23.2 (14.7-36.6)
19.6 (7.74-49.6)
11.5 (5.81-22.9)
8.61 (4.85-15.3)
32.8 (21.7-49.6)
18.6 (6.44-53.6)
15.6 (5.37-45.3)
134 (88.8-203)*
ND
100 (63.4-158)*
ND
3a
3b
3c
4a
4b
5a
5b
5c
55.0 (36.0-84.2)*
62.6 (44.7-87.7)*
44.9 (32.4-62.2)*
33.9 (23.1-49.8)*
85.9 (71.5-100)*
65.2 (43.1-98.6)*
31.4 (15.8-62.4)*
40.6 (30.1-54.9)*
39.6 (22.9-68.7)*
38.1 (28.2-58.5)*
31.0 (21.7-44.3)*
65.2 (43.1-98.6)*
85.9 (71.5-103)*
16.3 (6.87-38.7)*
a
All data were calculated according to the method of Litchfield and Wilcoxon.27 At least 32 animals were used to calculate each ED50
b
value. AMPA was administered icv at the CD97 for either clonus (9.7 nmol) or forelimb tonic extension (11.7 nmol) 30 min after injection
of tested compounds. c KA was administered sc at the CD97 (32 mg/kg) 15 min after injection of tested compounds. Significant differences
d
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 as *p < 0.01; ND, not determined.
the therapeutic index (TI) of all derivatives was always
more favorable than that of compound 1 (Table 1).
Binding experiments, carried out on crude cortical
synaptic membranes prepared from the rat brain,
showed that derivatives 4b and 5c (100 µM) failed to
displace [3H]spiperone from dopamine and 5-hydroxy-
tryptamine1 receptors (5-HT1); [3H]ketanserin from
5-hydroxytryptamine2 receptors (5-HT2); [125I]pindolol
from â-adrenergic receptors; [3H](RS)-3-(2-carboxypip-
erazin-4-yl)propyl-1-phosphonic acid ([3H]CPP) and [3H]-
dizocilpine 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/KA re-
ceptors; mixture of [3H](1S,3R)-ACPD and [3H](1R,3S)-
ACPD from metabotropic glutamate receptors. Due to
the lack of activity at dopamine, serotonin, noradrena-
line, NMDA, glycine site on NMDA, and metabotropic
glutamate receptors, it is conceivable to assume that
these new series of 2,3-benzodiazepines act as noncom-
petitive AMPA antagonists. Nevertheless, since no
reliable ligand-binding assay has been reported for
noncompetitive agonists and antagonists affecting the
AMPA receptor complex,14 our data do not exclude an
interaction at other sites involved in the generation or
expression of seizures.
receptors. Application of 100 µM KA induced an inward
current that is reduced by the coapplication of KA and
100 µM 4b (Figure 3). The degree of block of the peak
currents produced by each antagonist 3c, 4b, and 5c
(100 µM), expressed as the percent of reduction of the
KA currents (100%), is reported in Table 5. On the basis
of the structural similarity of 3c, 4b, and 5c with GYKI
53655 (2), it is reasonable to assess that the reduction
of the current is due to the selective block of the AMPA-
mediated component, while the remaining fast desen-
sitizing current results from the activation of the KA
receptors. The extent of the current reduction was
slightly higher for 3c than for 4b and 5c. The higher
activity in the in vivo test systems of 4b and 5c with
respect to 3c is likely due to their higher in vivo
bioavailability. Since GYKI 52466 potency has been
shown to be different at diverse AMPA receptor sub-
types,15 it is also possible that 4b and 5c show a
preferential activity at the level of specific AMPA
receptor subtypes involved in the genesis of seizure.
The anticonvulsant activity of compounds 4 and 5 was
effective at doses which did not cause sedation and
ataxia, in agreement with several studies which showed
an anticonvulsant activity of AMPA receptor antago-
nists at doses that do not affect behavior and learning
processes.16 It is noteworthy that the present compounds
4 and 5 cause marginal motor impairment in the rotarod
test at variance of 1; some of them possess TI values
approximately twice that of 1 (Table 1).
Block of KA-evoked current by 3c, 4b, and 5c was
assessed in whole-cell voltage clamp recording from
cerebellar granule neurons. KA elicits a current that is
mediated by the activation of both AMPA and KA
In conclusion, the present results indicate that re-

4418 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grasso et al.
F igu r e 2. Anticonvulsant effects of 5a -c and 1 against
seizures induced by AMPA in DBA/2 mice. Ordinate shows %
of response of clonic (A) or tonic (B) seizures; abscissa shows
the dose in µmol/kg ip. For the determination of each point,
10 animals were used.
F igu r e 1. Anticonvulsant effects of 4a ,b and 1 against
seizures induced by AMPA in DBA/2 mice. Ordinate shows %
of response of clonic (A) or tonic (B) seizures; abscissa shows
the dose in µmol/kg ip. For the determination of each point,
10 animals were used.
placement of the carbonyl group of 7,8-methylenedioxy-
4H-2,3-benzodiazepin-4-ones with the bioisosteric thio-
carbonyl moiety and introduction of a methylcarbamoyl
group at N-3 bring about an increase in anticonvulsant
potency. Derivative 4b, in analogy to that observed with
the analogues of GYKI 52466, shows ED50 values
against MES- and KA-induced seizures 2-3-fold higher
than parent compound 3c. Nevertheless, such an in-
crease was observed in the in vivo tests and not in the
electrophysiological experiments. Interestingly, deriva-
tive 4b is provided with a longer-lasting anticonvulsant
activity and a lower toxicity. As a consequence, 1-(4-
aminophenyl)-3,5-dihydro-3-methylcarbamoyl-7,8-meth-
ylenedioxy-4H-benzodiazepin-4-one (4b) may become a
useful tool in the mapping of the AMPA/KA receptor
complex.
F igu r e 3. Representative trace of the inward current evoked
by the application of 100 µM KA and its reduction in the
presence of compound 4b (100 µM). The cells were voltage-
clamped at -60 mV. Bars show the duration of drug applica-
tion.
Exp er im en ta l Section
Gen er a l P r oced u r e for th e Syn th esis of 3-Alk ylca r -
ba m oyl-1-(3- or 4-a m in op h en yl)-3,5-d ih yd r o-7,8-m eth yl-
en ed ioxy-4H-ben zod ia zep in -4-on es 4a -h . To a solution of
3d or 3e (0.5 g, 1.54 mmol) in CH2Cl2 (100 mL) were added
triethylamine (2 mL, 14.4 mmol) and the suitable isocyanate
(7.7 mmol). The reaction mixture was stirred at room temper-
ature for 36 h and then concentrated in vacuo. The resulting
residue was purified by column chromatography with CHCl3/
EtOAc (70:30) as eluant. The subsequent hydrogenation was
carried out at atmospheric pressure by adding 5% Pd/C (50
mg) to a methanol solution (40 mL) of the nitro derivative.
The mixture was shaken under hydrogen for 3 h and the Pd/C
was filtered out. The solvent was removed in vacuo and the
Ch em istr y. Melting points were determined on a Kofler
hot stage apparatus and are uncorrected. Elemental analyses
were carried out on a Carlo Erba 1106 elemental analyzer for
C, H, and N, 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
recorded in CDCl3 by means of a Varian Gemini-300 spec-
trometer. Chemical shifts were expressed in δ (ppm) relative
to TMS as internal standard, and coupling constants (J ) are
in Hz. All exchangeable protons were confirmed by addition
of D2O.

Benzodiazepine AMPA/ Kainate Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4419
Ta ble 5. Reduction of KA-Evoked Current Induced by 1, 2, 3c,
4b, and 5ca
2H, OCH2O), 6.68 (d, 2H, J ) 8.7, H-3′,5′), 6.74 (s, 1H, H-9),
6.86 (s, 1H, H-6), 7.56 (d, 2H, J ) 8.7, H-2′,6′), 8.72 (bs, 1H,
NH). Anal. (C21H22N4O4) C, H, N.
compd
% reductionb (mean ( SE)
Gen er a l P r oced u r e for th e Syn th esis of 1-Ar yl-3,5-
d ih yd r o-7,8-m et h ylen ed ioxy-4H -2,3-b en zod ia zep in e-4-
th ion es 5a -c. A solution of 3a -c (1 mmol) and Lawesson’s
reagent (0.22 g, 0.55 mmol) in dry toluene (50 mL) was heated
to reflux for 2 h. The solution was then cooled at room
temperature and filtered. The toluene was removed in vacuo,
the residue was purified by column chromatography with
EtOAc/CCl4 (70:30) as eluant and recrystallized from EtOAc
to give 5a -c.
3,5-Dih yd r o-7,8-m eth ylen ed ioxy-1-p h en yl-4H-2,3-ben -
zod ia zep in e-4-th ion e (5a ): mp 201-203 °C (0.25 g, 85%);
1H NMR 3.88 (bs, 2H, CH2-5), 6.04 (s, 2H, OCH2O), 6.61 (s,
1H, H-9), 6.88 (s, 1H, H-6), 7.41-7.63 (m, 5H, Ar), 9.91 (bs,
1H, NH). Anal. (C16H12N2O2S) C, H, N.
1
2
3c
4b
5c
73 ( 3
87 ( 3
79 ( 4
58 ( 4
66 ( 6
a
KA and compounds 1, 2, 3c, 4b, and 5c were tested at 100
µM. Each value is the mean ( SE of at least 10 cells.
b
resulting residue was purified by column chromatography with
CHCl3/MeOH (95:5) as eluant to give 4a -h . All compounds
were recrystallized from EtOAc.
1-(3-Am in op h en yl)-3,5-d ih yd r o-3-m et h ylca r b a m oyl-
7,8-m et h ylen ed ioxy-4H-b en zod ia zep in -4-on e (4a ): mp
137-140 °C (0.43 g, 80%); 1H NMR 2.92 (d, 3H, J ) 4.5, CH3),
3.52 (s, 2H, CH2-5), 3.82 (bs, 2H, NH2), 6.05 (m, 2H, OCH2O),
6.71 (s, 1H, H-9), 6.81 (dd, 1H, J ) 1.6 and 8.0, H-4′), 6.86 (s,
1H, H-6), 6.88 (dd, 1H, J ) 1.6 and 8.0, H-6′), 7.18 (t, 1H, J )
8.0, H-5′), 7.24 (s, 1H, H-2′), 8.62 (bs, 1H, NH). Anal.
(C18H16N4O4) C, H, N.
1-(4-Am in op h en yl)-3,5-d ih yd r o-3-m et h ylca r b a m oyl-
7,8-m eth ylen ed ioxy-4H-ben zod ia zep in -4-on e (4b): mp
140-143 °C (0.35 g, 65%); 1H NMR 2.91 (d, 3H, J ) 4.7, CH3),
3.49 and 3.54 (dd, 2H, J ) 12.4, CH2-5), 3.98 (bs, 2H, NH2),
6.05 (m, 2H, OCH2O), 6.68 (d, 2H, J ) 8.7, H-3′,5′), 6.73 (s,
1H, H-9), 6.86 (s, 1H, H-6), 7.56 (d, 2H, J ) 8.7, H-2′,6′), 8.66
(bs, 1H, NH). Anal. (C18H16N4O4) C, H, N.
1-(3-Am in op h en yl)-3,5-d ih yd r o-7,8-m et h ylen ed ioxy-
4H-2,3-ben zod ia zep in e-4-th ion e (5b): mp 122-124 °C (0.23
1
g, 75%); H NMR 3.87 (bs, 2H, CH2-5), 6.03 (s, 2H, OCH2O),
6.64 (s, 1H, H-9), 6.81(d, 1H, J ) 7.8, H-6′), 6.86 (s, 1H, H-6),
6.89 (d, 1H, J ) 7.8, H-4′), 6.97 (s, 1H, H-2′), 7.20 (t, 1H, J )
7.8, H-5′), 8.45 (bs, 1H, NH). Anal. (C16H13N3O2S) C, H, N.
1-(4-Am in op h en yl)-3,5-d ih yd r o-7,8-m et h ylen ed ioxy-
4H-2,3-ben zod ia zep in e-4-th ion e (5c): mp 138-140 °C (0.24
1
g, 77%); H NMR 3.84 (bs, 2H, CH2-5), 6.03 (s, 2H, OCH2O),
6.68 (s, 1H, H-9), 6.86 (s, 1H, H-6), 6.68 (d, 2H, J ) 8.3, H-3′,5′),
7.43 (d, 2H, J ) 8.3, H-2′,6′), 9.91 (bs, 1H, NH). Anal.
(C16H13N3O2S) 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.17 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 lipo-
philicity.
Testin g of An ticon vu lsa n t Activity Aga in st Au d io-
gen ic Seizu r es in DBA/2 Mice. All experiments were
performed with DBA/2 mice which are genetically susceptible
to sound-induced seizures.18 Male DBA/2 mice (8-12 g, 22-
25 days old) were purchased from Charles River (Calco, Como,
Italy). Groups of 10 mice 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
30% dimethyl sulfoxide (DMSO) and 70% 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 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.19 The time course of the anticonvulsant
action of 3 and 5 was determined following the administration
of 33 µmol/kg of each benzodiazepine derivative to groups of
10 mice for each time. The animals were tested for sound-
induced seizure responses at 5-180 min after drug adminis-
tration.
1-(3-Am in op h en yl)-3,5-d ih yd r o-3-eth ylca r ba m oyl-7,8-
m eth ylen ed ioxy-4H-ben zod ia zep in -4-on e (4c): mp 110-
112 °C (0.39 g, 70%); 1H NMR 1.20 (t, 3H, J ) 7.4, CH3), 3.38
(m, 2H, CH2), 3.51 (s, 2H, CH2-5), 3.80 (bs, 2H, NH2) 6.05 (m,
2H, OCH2O), 6.71 (s, 1H, H-9), 6.88 (dd, 1H, J ) 1.5 and 8.0,
H-4′), 6.86 (s, 1H, H-6), 6.88 (dd, 1H, J ) 1.5 and 8.0, H-6′),
7.18 (t, 1H, J ) 8.0, H-5′), 7.25 (s, 1H, H-2′), 8.73 (bs, 1H, NH).
Anal. (C19H18N4O4) C, H, N.
1-(4-Am in op h en yl)-3,5-d ih yd r o-3-eth ylca r ba m oyl-7,8-
m eth ylen ed ioxy-4H-ben zod ia zep in -4-on e (4d ): mp 111-
114 °C (0.46 g, 81%); 1H NMR 1.20 (t, 3H, J ) 7.4, CH3), 3.37
(m, 2H, CH2), 3.48 and 3.53 (dd, 2H, J ) 12.5, CH2-5), 4.00
(bs, 2H, NH2), 6.05 (m, 2H, OCH2O), 6.68 (d, 2H, J ) 8.4,
H-3′,5′), 6.73 (s, 1H, H-9), 6.86 (s, 1H, H-6), 7.56 (d, 2H, J )
8.4, H-2′,6′), 8.69 (bs, 1H, NH). Anal. (C19H18N4O4) C, H, N.
1-(3-Am in op h en yl)-3,5-d ih yd r o-7,8-m eth ylen ed ioxy-3-
p r op ylca r ba m oyl-4H-ben zod ia zep in -4-on e (4e): mp 116-
118 °C (0.42 g, 72%); 1H NMR 0.94 (t, 3H, J ) 7.3, CH3), 1.59
(m, 2H, CH2), 3.30 (m, 2H, CH2), 3.52 (s, 2H, CH2-5), 3.80 (bs,
2H, NH2), 6.04 (m, 2H, OCH2O), 6.71 (s, 1H, H-9), 6.80 (dd,
1H, J ) 1.4 and 7.9, H-4′), 6.86 (s, 1H, H-6), 6.88 (dd, 1H, J )
1.4 and 7.9, H-6′), 7.18 (t, 1H, J ) 7.9, H-5′), 7.24 (s, 1H, H-2′),
8.73 (bs, 1H, NH). Anal. (C20H20N4O4) C, H, N.
1-(4-Am in op h en yl)-3,5-d ih yd r o-7,8-m eth ylen ed ioxy-3-
p r op ylca r ba m oyl-4H-ben zod ia zep in -4-on e (4f): mp 135-
137 °C (0.41 g, 70%); 1H NMR 0.94 (t, 3H, J ) 7.4, CH3), 1.58
(m, 2H, CH2), 3.27 (m, 2H, CH2), 3.48 and 3.54 (dd, 2H, J )
12.4, CH2-5), 3.97 (bs, 2H, NH2), 6.05 (m, 2H, OCH2O), 6.68
(d, 2H, J ) 8.7, H-3′,5′), 6.74 (s, 1H, H-9), 6.86 (s, 1H, H-6),
7.56 (d, 2H, J ) 8.7, H-2′,6′), 8.74 (bs, 1H, NH). Anal.
(C20H20N4O4) C, H, N.
1-(3-Am in op h en yl)-3-bu tylca r ba m oyl-3,5-d ih yd r o-7,8-
m eth ylen ed ioxy-4H-ben zod ia zep in -4-on e (4g): mp 176-
178 °C (0.39 g, 65%); 1H NMR 0.93 (t, 3H, J ) 7.4, CH3), 1.35
(m, 2H, CH2), 1.49 (m, 2H, CH2), 3.34 (m, 2H, CH2), 3.52 (s,
2H, CH2-5), 3.90 (bs, 2H, NH2), 6.05 (m, 2H, OCH2O), 6.71 (s,
1H, H-9), 6.81 (dd, 1H, J ) 1.5 and 8.0, H-4′), 6.86 (s, 1H, H-6),
6.88 (dd, 1H, J ) 1.5 and 8.0, H-6′), 7.18 (t, 1H, J ) 7.8, H-5′),
7.25 (s, 1H, H-2′), 8.73 (bs, 1H, NH). Anal. (C21H22N4O4) C, H,
N.
1-(4-Am in op h en yl)-3-bu tylca r ba m oyl-3,5-d ih yd r o-7,8-
m eth ylen ed ioxy-4H-ben zod ia zep in -4-on e (4h ): mp 173-
175 °C (0.41 g, 67%); 1H NMR 0.93 (t, 3H, J ) 7.4, CH3), 1.35
(m, 2H, CH2), 1.39 (m, 2H, CH2), 3.33 (m, 2H, CH2), 3.48 and
3.53 (dd, 2H, J ) 12.4, CH2-5), 3.97 (bs, 2H, NH2), 6.05 (m,
MES 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

4420 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Grasso et al.
et al.20 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.
motor movements continuously for 2 min on a rotarod, 3 cm
in 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.26 The ability of the mice to remain on the rotarod
was tested 30 min after administration of various compounds.
St a t ist ica 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 audiogenic
seizure or seizures induced by MES, PTZ, AMPA, or KA were
determined for each dose of compound administered, and
dose-response curves were fitted using a computer program
by the method of Litchfield and Wilcoxon.27 The relative
anticonvulsant activities were determined by comparison of
respective ED50 values. The TD50 values were estimated using
the method of Litchfield and Wilcoxon.27 The relative activities
were determined by comparison of respective TD50 values. For
the binding experiments IC50 values were determined by the
nonlinear curve-fitting program based on Ligand.28 Statistical
significance between control and test groups of data means
was tested using a two-tailed Student’s t-test. Electrophysi-
ological data were analyzed using the software Clampex (Axon
Instrument). Results are expressed as mean ( SE. Origin
(Microcal Software, Northampton, MA) was used for figure
preparation and statistical analysis.
P TZ-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 pretreated with vehicle or drug 45
min before sc administration of PTZ. 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 30% DMSO
and 70% 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 PTZ seizure threshold.21
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;22 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.
P r etr ea tm en t w ith An ir a ceta m . The icv microinjection
of aniracetam was performed according to experimental pro-
cedures previously described for AMPA microinjection.21 The
dose of aniracetam (50 nmol icv) was administered 60 min
before auditory stimulation or 30 min before each compound
in DBA/2 mice.
KA-In d u ced Seizu r es in Sw iss Mice. KA was adminis-
tered sc at a dose of 32 mg/kg (previously determined CD97
value) 15 min after ip administration of the 2,3-benzodiazepine
derivative. Animals showing 5 s or more of clonic activity were
scored as not protected according to Donevan et al.6 The period
of observation was 60 min.
Ack n ow led gm en t. This work was supported by
CNR, 98.01940.CT03 (Rome, Italy).
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neurons were prepared from 7-8 days old Sprague-Dawley
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of 106 cells/mL on 35-mm Falcon dishes coated with poly-L-
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configuration.24 Electrodes were pulled from borosilicate glass
on a vertical puller and had a resistance of 5-7 MΩ when filled
with KCl internal solution. Currents were amplified with an
Axopatch 1D amplifier, filtered at 5 kHz, and digitized at 10
kHz by using pClamp software. Intracellular solution con-
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glucose 5, sucrose 20, pH 7.4 with NaOH. KA was purchased
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a gift from Lilly Research Lab. GYKI 52466, GYKI 53655, 3c,
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concentration (<1%) in extracellular medium. KA was dis-
solved in the extracellular solution. All drugs were applied
directly by gravity through a Y-tube perfusion system.25
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the methods previously described by us.8
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