Novel potent AMPA/kainate receptor antagonists: Synthesis and anticonvulsant activity of a series of 2-[(4-alkylsemicarbazono)-(4-amino-phenyl)methyl]-4, 5-methylenedioxyphenylacetic Acid alkyl esters

Article abstract of DOI:10.1021/jm020863y

In this paper we describe the synthesis of a series of novel 2-[(4-alkylsemicarbazono)-(4-aminophenyl)-methyl]-4, 5-methylenedioxyphenylacetic acid alkyl esters (10-19) carrying an alkylsemicarbazono moiety at a benzylic site. The influence of this group on the biological activity was evaluated by testing the corresponding derivatives 20-22 in which the 4-alkylsemicarbazono moiety was removed (compound 20) or its alkylureido portion shifted at position 1 (compounds 21-22). Furthermore, the involvement of the 4-aminobenzyl moiety in the anticonvulsant activity was evaluated by testing derivative 23. The anticonvulsant activity of all compounds was assayed against audiogenic seizures induced in DBA/2 mice. Within this series of derivatives, 2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4, 5-methylenedioxyphenylacetic acid methyl ester (10) proved to be the most active compound. It displayed a potency 5-fold higher than that shown by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonist. Compound 10 was also effective in suppressing seizures induced in Swiss mice by maximal electroshock (MES) or pentylene-tetrazole (PTZ). Furthermore, it antagonized in vivo seizures induced by icv administration of AMPA or kainate (KA). Using the patch-clamp technique in primary cultures of granule neurons we tested compounds 10 and 21 for their ability to modulate currents evoked by KA and 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA). These two derivatives reduced KA and ATPA currents to a larger extent than that shown by reference compound 1. Compounds 10 and 21 were also able to reduce neuronal cell death induced by the application of KA (100 μM).

Full text of DOI:10.1021/jm020863y

J . Med. Chem. 2002, 45, 4433-4442
4433
Novel P oten t AMP A/Ka in a te Recep tor An ta gon ists: Syn th esis a n d
An ticon vu lsa n t Activity of a Ser ies of 2-[(4-Alk ylsem ica r ba zon o)-(4-a m in o-
p h en yl)m eth yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid Alk yl Ester s
Nicola Micale,† Maria Zappala`,† Silvana Grasso,*,† Giulia Puja,§ Giovambattista De Sarro,| Guido Ferreri,|
Angela De Sarro,‡ Lucio Toma, and Carlo De Micheli#
Dipartimento Farmaco-Chimico and Istituto di Farmacologia, Universita` di Messina, Italy, Dipartimento di Scienze
Farmaceutiche, Universita` di Modena, Italy, Dipartimento di Medicina Sperimentale e Clinica, Universita` di Catanzaro, Italy,
Dipartimento di Chimica Organica, Universita` di Pavia, Italy, and Istituto di Chimica Farmaceutica, Universita` di Milano,
Italy
Received February 25, 2002
In this paper we describe the synthesis of a series of novel 2-[(4-alkylsemicarbazono)-(4-
aminophenyl)-methyl]-4,5-methylenedioxyphenylacetic acid alkyl esters (10-19) carrying an
alkylsemicarbazono moiety at a benzylic site. The influence of this group on the biological
activity was evaluated by testing the corresponding derivatives 20-22 in which the 4-alkylsemi-
carbazono moiety was removed (compound 20) or its alkylureido portion shifted at position 1
(compounds 21-22). Furthermore, the involvement of the 4-aminobenzyl moiety in the
anticonvulsant activity was evaluated by testing derivative 23. The anticonvulsant activity of
all compounds was assayed against audiogenic seizures induced in DBA/2 mice. Within this
series of derivatives, 2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5-methylenedi-
oxyphenylacetic acid methyl ester (10) proved to be the most active compound. It displayed a
potency 5-fold higher than that shown by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-
2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive 2-amino-3-(3-hydroxy-5-
methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonist. Compound 10 was also effective
in suppressing seizures induced in Swiss mice by maximal electroshock (MES) or pentylene-
tetrazole (PTZ). Furthermore, it antagonized in vivo seizures induced by icv administration of
AMPA or kainate (KA). Using the patch-clamp technique in primary cultures of granule neurons
we tested compounds 10 and 21 for their ability to modulate currents evoked by KA and
2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA). These two derivatives
reduced KA and ATPA currents to a larger extent than that shown by reference compound 1.
Compounds 10 and 21 were also able to reduce neuronal cell death induced by the application
of KA (100 µM).
In tr od u ction
be relatively well tolerated and devoid of psychotomi-
metic effects.3
Hyperactivity of ionotropic glutamate receptors, clas-
sified as N-methyl-D-aspartic acid (NMDA), 2-amino-3-
(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA),
and kainic acid (KA), is believed to be implicated in
neuronal damage in humans suffering from acute
degenerative disorders such as stroke and epilepsy, or
chronic degenerative diseases including amyotrophic
lateral sclerosis, Huntington’s chorea, and Parkinson’s
and Alzheimer’s diseases.1 Consistent with this hypoth-
esis, competitive and noncompetitive antagonists of
these receptors are attractive therapeutic targets.2 To
date, interest in this area seems to be focused on
antagonists acting selectively on AMPA and KA recep-
tors due to their effectiveness in the treatment of
epilepsy and cerebral ischaemia, and because, at vari-
ance with NMDA receptor antagonists, they appear to
1-(4-Aminophenyl)-4-methyl-7,8-methylenedioxy-5H-
2,3-benzodiazepine (1, GYKI 52466) (Chart 1), the
prototype of noncompetitive AMPA receptor antagonists
endowed with anticonvulsant and neuroprotective prop-
erties,4 induced growing interest on 2,3-benzodiazepine
derivatives. A significant improvement in the pharma-
cological profile of GYKI 52466 was obtained by ap-
pending an alkylcarbamoyl group at N-3 of the benzo-
diazepine ring.5 As a matter of fact, the 3-N-methyl-
carbamoyl derivative 2 (GYKI 53655, LY 300168) (Chart
1) emerged as the most potent compound among this
series of derivatives.6
As part of a program aimed at identifying potent and
selective AMPA receptor antagonists, we7 and other
authors8 previously reported investigations on a series
of 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzo-
diazepin-4-ones where the iminohydrazone portion of 1
was replaced by the iminohydrazide moiety. In this
context we noticed that derivative 3 (Chart 1) was
roughly 2-fold more potent than 1 and was provided
with a better protective index. Taking into account
modifications previously made5 on analogues of GYKI
52466, we prepared a number of 3-N-alkylcarbamoyl-
derivatives (e.g., 4-7) (Chart 1). The results indicated
* 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, Universita` di Messina.
‡ Istituto di Farmacologia, Universita` di Messina.
§ Universita` di Modena.
| Universita` di Catanzaro.
Universita` di Pavia.
Universita` di Milano.
#
10.1021/jm020863y CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/24/2002

4434 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Micale et al.
Ch a r t 1
Sch em e 1a
a
Reagents: (a) CH2Cl2, Et3N, RNCO, rt, 36 h; (b) anhydrous
alcohols, SnCl2, 80 °C, 2 h.
Encouraged by these results, we designed and biologi-
cally evaluated a series of alkyl esters of 4,5-methylen-
dioxyphenylacetic acids 10-19 bearing a 4-alkylsemi-
carbazono moiety at position 2, that may be envisaged
as “open models” of reference compounds 4-7 (Chart
1). The influence of this group on the biological activity
was evaluated by testing the corresponding derivatives
20-22, in which the 4-alkylsemicarbazono moiety was
removed (compound 20) or its alkylureido portion shift-
ed at position 1 (compounds 21-22). Furthermore, the
involvement of the 4-aminobenzyl moiety in the anti-
convulsant activity was evaluated by testing derivative
23.
that the introduction of a methylcarbamoyl group at
N-3, i.e., compound 4, increased the anticonvulsant
potency.9 Worth noting, derivative 4 was also provided
with a longer-lasting anticonvulsant activity.
In our ongoing studies on the structure-activity rela-
tionships (SAR) of 2,3-benzodiazepine derivatives,10 we
have recently prepared11 a number of 1,2,3,5-tetrahydro-
2,3-benzodiazepine derivatives, i.e., 8, as well as a series
of novel phthalazin-1(2H)-ones, i.e., 9, to check the influ-
ence of both the 1,2-azomethine moiety of the diazepine
nucleus and the size of the heterocyclic ring on the anti-
convulsant activity. The results showed that some deriv-
atives, 8, were provided with a noteworthy anticonvul-
sant activity which was longer-lasting when compared
to that shown by the corresponding unsaturated ana-
logues. On the other hand, the activity of the phthal-
azino derivatives 9 was strictly dependent upon the
alkyl group of the carbamoyl moiety appended at N-2.
The peak value was reached with the n-butyl group.
Ch em istr y
The synthesis of 2-[(4-alkylsemicarbazono)-(4-ami-
nophenyl)-methyl]-4,5-methylenedioxyphenylacetic acid
alkyl esters (10-19) was accomplished according to the
reaction sequence reported in Scheme 1.
3,5-Dihydro-7,8-methylenedioxy-1-(4-nitrophenyl)-4H-
2,3-benzodiazepin-4-one, 24, used as the reagent, was
prepared according to the procedure previously re-
ported.8 It was treated with an excess of the appropriate
alkyl isocyanate in the presence of triethylamine to yield
the corresponding 3-N-alkylcarbamoyl derivatives 25-
28. If the reduction of intermediates 25-28 was carried
out under mild conditions, i.e., catalytic hydrogenation,

Novel Potent AMPA/ Kainate Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4435
F igu r e 1. 3D Plot of a representative conformation of stereoisomers 10 and 29.
5% Pd/C-methanol at room temperature, the corre-
sponding amino derivatives 4-7 were obtained in good
yields and pure form.9 Conversely, if the same reaction
was conducted under harsh conditions, i.e., tin(II)
chloride in various alcohols at reflux for 2 h, compounds
10-19 were obtained in reasonable yield. The expected
reduction of the nitro group to the amino moiety was
accompanied by the cleavage of the heterocyclic ring.
Conceivably, the nucleophilic attack of the alcohol at
C-4, which caused the cleavage of the C-N bond, was
catalyzed by tin(II) chloride, the Lewis acid. As a matter
of fact, heating at reflux of a methanol solution of both
4 and 25 did not cause the cleavage of the heterocyclic
ring, but resulted in the removal of their side-chain.
of fact, 1H NMR spectra of derivatives 10 and 29,
recorded both in acetone-d6 and DMSO-d6, showed the
splitting of some signals, thus confirming the above-
reported observations. The two CH2 groups, i.e., the one
in the R position in respect to the ester function and
the one of the dioxole ring, appeared as singlets in
derivative 29 and as AB systems or multiplets in its
Z-form (10). These data testify that the geminal hydro-
gens are diastereotopic in 10 because of the hindered
rotation around the single bond connecting the semi-
carbazono moiety to C-2. The same does not hold true
for derivative 29 because of the much easier rotation
around the same single bond.
NOESY experiments, performed in DMSO-d6, con-
firmed the proposed structural assignment. In fact, a
strong cross-peak between NH-2 of the semicarbazono
moiety and aromatic 2′ and 6′ protons was evidenced
in derivative 29 and was not detected in its stereoisomer
10. These experimental findings support the value of
the distance between NH-2 and the nearest of the two
2′ and 6′ protons found in various conformations of 29
(2.4-2.6 Å); a value quite different from that observed
in derivative 10 (4.4-4.8 Å). In Figure 1 is depicted a
representative conformation of stereoisomers 10 and 29.
Conversely, the NH-2 proton gave a weak cross-peak
with H-3 in derivative 10 but not in its isomer 29; once
again supporting the values of the corresponding dis-
tances (2.9-3.6 Å for 10 versus 4.5-4.7 Å for 29)
calculated for their most stable conformations.
The nucleophilic attack of the alcohol to the carbonyl
moiety of bicyclic derivatives 25-28 yielded the corre-
sponding monocyclic analogues 10-19 as single isomers.
During the synthesis of (-)-3-acetyl-1-(4-aminophenyl)-
3,4-dihydro-4-methyl-7,8-methylenedioxy-5H-2,3-benzo-
diazepine (LY300164) some authors prepared interme-
diate hydrazones similar to the present ones and noticed
that they were composed by an inseparable mixture of
E/ Z isomers.12 A chloroform solution of 10 left aside for
two weeks at room temperature gave a 70:30 mixture
of 10 and 29; the two isomers were easily separated by
a silica gel column chromatography. HPLC-MS analy-
sis of the mixture showed that 10 and 29 were isomers
and they possessed the same fragmentation pattern (see
Experimental Section). The stereochemistry around the
CdN bond of semicarbazono derivatives 10-19 was
investigated on the couple of stereoisomers, 10 and 29,
Compound 20 was synthesized according to the reac-
tion sequence reported in Scheme 2. The reduction of
the keto group of 308 to yield 31 was efficiently achieved
with sodium cyanoborohydride in the presence of 3
equivalents of boron trifluoride diethyl etherate.14 The
nitro ester 31 was transformed into the corresponding
amino derivative 20 under standard conditions. Ester
31 was also hydrolyzed to acid 32 and subsequently
transformed into the corresponding acyl chloride with
a standard methodology. Such an intermediate was not
characterized but directly reacted with N-methylurea
to afford compound 33 in reasonable yield. Reduction
of the nitro group of 33 was accomplished with Raney-
Ni/ammonium formate and gave final derivative 1-[2-
1
using a combination of modeling studies and H NMR
spectroscopy. The conformational preferences of both the
compounds 10 and 29 were evaluated at the semiem-
pirical AM1 level.13 Both the compounds showed a large
conformational freedom with some relevant differences.
The rotation around the single bond connecting the
semicarbazono moiety to C-2 appeared to be easier for
the E-isomer (29) than for the corresponding Z-form
(10). The energy barrier, corresponding to the planar
arrangement of the groups, was about 6-9 kcal/mol for
29 and about 18-22 kcal/mol for 10, making derivative
10 a chiral molecule in the NMR time scale. As a matter

4436 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Micale et al.
Sch em e 2a
following structural modifications: (i) the size of the
alkyl group appended at the semicarbazono moiety; (ii)
the removal of the semicarbazono moiety or the shift of
its alkylureido portion to position 1; (iii) the bulkiness
of the ester alkyl group; and (iv) the presence of the
4-aminobenzyl group. The data will be discussed in
comparison to those previously reported by us9 for the
corresponding bicyclic derivatives 3-7, taken as models.
Among the compounds examined, derivatives 10-12,
14, 21, and 29 possess an anticonvulsant activity equal
to or higher than that displayed by 1. In particular,
compounds 10 and 21 are 5- and 4-fold more potent than
1 with ED50 values of 7.87 and 9.28 µmol/kg, respec-
tively. It is worth noting that 10 carries the smallest
alkyl group on both the semicarbazono and ester
moieties. The same trend was previously noticed in the
series of 3-N-alkylcarbamoyl-2,3-benzodiazepines where
the methyl group gave the peak value (e.g., 4 vs 5-7,
Table 1). Compound 29, the E-form of 10, possesses a
noteworthy activity even if slightly lower than that of
10. In any case, we cannot exclude an in vivo intercon-
version among stereoisomers 10 and 29. An increase in
the bulkiness of the alkyl group at the ester side
produces a decrease in activity, i.e., 10 > 11-13 and
14 > 15-17 (Table 1). The same trend holds true for
the alkyl group appended at the semicarbazono moiety,
i.e., 10 > 14, 18-19 (Table 1). These observations seem
to indicate that lipophilicity plays a pivotal role in
determining the anticonvulsant activity of compounds
10-19. Usually, an increase of lipophilicity reduces the
anticonvulsant activity, in analogy to the trend previ-
ously observed in the set of bicyclic derivatives, i.e., 4
> 5-7. The sole exception is represented by compounds
21 and 22 where the higher lipophilicity of 21 (Rm
-0.570 for 21 vs -0.689 for 22) is reflected in a higher
anticonvulsant activity (ED50 9.28 for 21 vs 39.5 for 22).
The different hybridization of the carbon bearing the
aryl group could modify the spatial arrangement of such
a moiety. The lack of anticonvulsant activity of 23 could
be attributed to the absence of the p-aminobenzyl group
which seems to be an essential feature in this series of
compounds since the most active noncompetitive AMPA
antagonists, provided with the 2,3-benzodiazepine
nucleus, bear the 4-aminophenyl moiety in position 1
of the bicyclic system.10 A comparison of the anticon-
vulsant activity displayed by compounds 10, 20, and 21
indicates that the methylsemicarbazono moiety plays
a role of utmost importance in determining the rank of
activity. As a matter of fact, the shift of its methylureido
portion from position 2 to position 1, i.e., on passing from
10 to 21, marginally influences the activity, whereas its
removal gives an inactive compound (20).
a
Reagents: (a) NaBH3CN, BF3OEt2, anhydrous THF, reflux,
16 h; (b) EtOH, Raney-Ni, ammonium formate; (c) H2O/THF, HCl
6 N, reflux, 9 h; (d) SOCl2; (e) methylurea, benzene, reflux, 6 h; (f)
KMnO4, acetone, rt, 3 h, Na2SO4.
(4-aminobenzyl)-4,5-methylenedioxyphenylacetyl]-3-me-
thylurea (21) in good yield. 1-[2-(4-Aminobenzoyl)-4,5-
methylenedioxyphenylacetyl]-3-methylurea (22) was
obtained through an oxidation of intermediate 33 with
potassium permanganate followed by a reduction of its
nitro group carried out under standard conditions
(Scheme 2). Finally, compound 23 was easily prepared
by coupling 3,4-methylenedioxyphenylacetyl chloride
with methylurea at reflux in benzene.
Resu lts a n d Discu ssion
Novel 2-[(4-alkylsemicarbazono)-(4-aminophenyl)-meth-
yl]-4,5-methylenedioxy-phenylacetic acid alkyl esters
(10-19), 2-(4-aminophenyl)-4,5-methylenedioxyphenyl-
acetic acid methyl ester (20), 1-[2-(4-aminobenzyl)-4,5-
methylenedioxyphenylacetyl]-3-methylurea (21), 1-[2-
(4-aminobenzoyl)-4,5-methylenedioxyphenylacetyl]-3-
methylurea (22), and 1-[2-(3,4-methylenedioxyphenyl-
acetyl)]-3-methylurea (23) were evaluated for their
anticonvulsant properties in DBA/2 mice, a strain gene-
tically susceptible to sound-induced seizures. This test
has been considered an excellent animal model for gen-
eralized epilepsy and for screening of new anticonvul-
sant drugs.15 Compounds 10-23 were administered
intraperitoneally (ip) at doses spanning the range 3.3-
200 µmol/kg, and their anticonvulsant properties, ex-
pressed as median effective dose (ED50) (Table 1), were
evaluated 30 min after injection.
No adverse side effects, such as sedation or ataxia,
were observed for novel compounds 10-23 and 29 at
the doses used to test their anticonvulsant activity, in
agreement with the results obtained with different
series’ of noncompetitive AMPA antagonists.6,7,9 It is
noteworthy that all compounds possess a protective
index (PI) higher than that shown by 1 (Table 1).
Because of its potent anticonvulsant activity in the
audiogenic seizure model, compound 10 was further
investigated and the results are herewith compared
with those of the related bicyclic derivatives 3-4 as well
as model compound 1.
The structure-activity relationships among this se-
ries of derivatives were examined in respect to the

Novel Potent AMPA/ Kainate Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4437
Ta ble 1. Anticonvulsant Activity of Compounds 1, 3-7, 10-23, and 29 Against Audiogenic Seizures in DBA/2 Mice, TD50 Values on
Locomotion Assessed by Rotarod Test, Relative Lipophilicity (Rm), and Calculated log P
ED50, µmol/kga
clonic phase
TD50, µmol/kg,a
locomotor deficit
PI,b
TD50/ED50
compd
tonic phase
Rm
log Pc
1
3
4
5
6
7
35.8 (24.4-52.4)
15.4 (10.1-23.5)
12.4 (6.44-23.8)
35.0 (18.5-66.3)
69.4 (36.2-133)
38.7 (21.2-70.8)
7.87 (4.68-13.2)
28.8 (16.9-49.1)
18.0 (10.8-30.2)
66.2 (43.1-101)
30.3 (24.4-37.8)
84.4 (64.3-110)
55.5 (44.3-69.5)
53.1 (39.1-72.0)
57.1 (41.5-78.5)
65.0 (54.7-77.2)
54.3 (42.9-68.9)
9.28 (4.82-17.8)
39.5 (29.4-53.0)
87.8 (63.4-121)
10.4 (5.22-20.8)
25.3 (16.0-40.0)
10.9 (4.60-24.6)
8.70 (4.61-16.4)
23.8 (13.4-42.1)
49.2 (25.9-93.4)
32.6 (18.2-58.4)
4.62 (2.47-8.61)
10.9 (5.76-20.8)
8.93 (4.40-18.1)
39.8 (28.8-55.1)
32.8 (19.3-29.5)
60.8 (44.9-82.2)
40.3 (28.7-52.6)
38.7 (28.5-52.6)
42.8 (24.5-74.7)
38.2 (23.8-61.4)
47.7 (38.7-58.9)
7.65 (3.79-46.3)
28.9 (17.9-46.3)
74.4 (41.9-132)
9.51 (5.09-17.7)
76.1 (47.5-122)
99.1 (72.4-135)
48.6 (31.4-54.6)
134 (70.7-255)
182 (95.1-350)
108 (82.2-143)
28.3 (19.0-42.3)
95.1 (65.6-137)
68.5 (41.5-112)
179 (125-256)
113 (84.6-152)
227 (174-229)
187 (143-249)
117 (93.9-145)
177 (137-228)
188 (164-216)
169 (154-214)
33.3 (18.8-19.0)
134 (102-174)
219 (159-303)
38.6 (26.9-53.4)
2.1
6.3
3.9
3.8
2.6
2.8
3.6
3.3
3.8
2.7
3.7
2.7
3.4
2.2
3.1
2.9
3.1
3.6
3.4
2.5
3.7
-0.502
-0.634
-0.703
-0.620
-0.564
-0.428
-0.682
-0.452
-0.364
-0.244
-0.560
-0.490
-0.254
-0.135
-0.463
-0.225
-0.456
-0.570
-0.689
-0.916
-0.583
3.51
2.58
1.99
2.41
2.77
3.34
2.46
2.89
3.24
3.81
2.89
3.31
3.67
4.24
3.24
3.81
2.68
2.23
1.82
1.09
2.99
10
11
12
13
14
15
16
17
18
19
20
21
22
23
29
a
All data were calculated according to the method of Litchfield and Wilcoxon.29 95% confidence limits are given in parentheses. At
b
least 32 animals administered ip were used to calculate each ED50 and TD50 value. PI, protective index, represents the ratio between
TD50 and ED50 (from the clonic phase of the audiogenic seizures). c Calculated with the XLOGP 2.0 program.
Ta ble 2. ED50 Values at Various Times Following Intraperitoneal Administration of Compounds 1, 3, 4, and 10
ED50, µmol/kg ((95% confidence limits),a clonic phase
compd
15 min
30 min
45 min
60 min
90 min
> 50
120 min
> 50
1
10.8
(7.11-16.4)
7.55
(4.08-14.0)
14.3
35.8
(24.4-62.4)
15.4**
(10.1-23.5)
12.4**
37.3
(27.2-52-1)
18.7**
(11.3-31.0)
12.1**
39.5
(29.6-52.7)
21.3*
(14.2-31.9)
13.0**
3
> 50
> 50
4
19.4**
40.8
(7.54-27.0)
24.1
(6.44-23.8)
7.87**
(7.28-20.2)
7.75**
(7.40-22.8)
10.2**
(7.96-47.5)
25.5*
(13.2-126)
36.8
10
(13.6-42.6)
(4.68-13.2)
(4.62-13.0)
(3.87-26.9)
(11.1-56.5)
(26.6-50.9)
a
Significant differences among compounds 1, 3, 4, and 10 were evaluated at the corresponding times and denoted as *p < 0.05 and **p
< 0.01 using the method of Litchfield and Wilcoxon.29
Ta ble 3. ED50 Values of 1, 3, 4, and 10 Against MES- and PTZ-Induced Seizures in Swiss Mice, and Against AMPA- and
KA-Induced Seizures in DBA/2 Mice
ED50 µmol/kga (( 95% confidence limits)
MES
PTZ
AMPAb
KAc
compd
tonus
clonus
tonus
57.5
clonus
40.5
1
35.7
68.3
27.8
(29.3-43.4)
32.1
(56.2-83.1)
71.8
(43.5-76.0)
37.6
(26.3-60.8)
27.0
(18.8-40.9)
19.6
3
(23.2-44.3)
18.6
(53.2-96.9)
16.3
(26.4-53.6)
18.6
(18.4-40.3)
10.3
(7.74-49.6)
8.61
4
(8.40-41.2)
15.7
(7.30-36.0)
14.7
(6.44-53.6)
13.9
(5.27-20.1)
8.9
(4.85-15.3)
16.6
10
(11.7-21.3)
(10.1-21.3)
(9.7-19.9)
(6.3-31.8)
(8.67-31.8)
a
All data were calculated according to the method of Litchfield and Wilcoxon.29 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 ip
injection of tested compounds. c KA was administered sc at the CD97 (32 mg/kg) 15 min after ip injection of tested compounds.
The time-course of the anticonvulsant activity of
derivative 10 is similar to that of 3-N-methylcarbamoyl
derivative 4, as they reach the maximum value of
protection between 30 and 45 min with a return to
control after 120 min from ip administration (Table 2).
On the contrary, model compounds 1 and 3 displayed
their maximum protection at 15 min from ip adminis-
tration and returned to control at 90 min. These results
suggest that the presence of the N-methylcarbamoyl
group is responsible for the longer-lasting activity of 4
and 10 with respect to 3.
As shown in Table 3, the tonic extension and the
clonic phase of the seizures induced in Swiss mice by
maximal electroshock (MES) and pentylenetetrazole
(PTZ), respectively, were significantly reduced at 45 min
after ip administration of compound 10. Compound 10
is definitely more effective than GYKI 52466 and 3 in
both tests and is equiactive with bicyclic derivative 4.

4438 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Micale et al.
Ta ble 4. Percentage of Reduction of KA- and ATPA-evoked
Currents Induced by 1, 3, 4, 10, and 21a
% reductionb (mean ( SE)
compd
KA
ATPA
1
3
4
10
21
47 ( 4
79 ( 4
58 ( 4
60 ( 1
54 ( 7
45 ( 7
94 ( 1
50 ( 3
75 ( 5
32 ( 5
a
KA, ATPA, compounds 1, 3, 4, 10, and 21 were tested at 100
b
µM. Each value is the mean ( SE of at least 10 cells.
To investigate the relationship between the anticon-
vulsant activity of 10 and its activity in non-NMDA
receptors, additional tests were performed (Table 3).
Compound 10 produced a dose-dependent protection
against seizures induced by the intracerebroventricular
(icv) administration of AMPA and subcutaneous (sc)
administration of KA. 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 3).
F igu r e 2. Protective effects of compounds 10 and 21 against
neuronal cell death induced by 100 µM KA for 3 h. Toxicity
experiment was performed on primary cultures of rat cortical
neurons at 8 DIV. Bar plot showing the % of cell viability
expressed in representative experiments. Bars represent mean
values ( SE from 8 determinations. Asterisks mean significant
differences (ANOVA p < 0.01) between KA alone and with 10
or 21.
The activity of derivatives 10 and 21 on KA- and
ATPA-evoked currents was assessed using the patch-
clamp technique in cerebellar neurons grown in primary
cultures. At variance of AMPA-response which is fast
desensitizing,16 KA elicits an inward nondesensitizing
current that is mediated by the activation of both AMPA
and KA receptors, whereas ATPA selectively activates
GluR5, a subtype of the KA receptors. KA- and ATPA-
evoked currents were reduced by the application of 10
and 21 (100 µM) to an extent that was comparable to
that of 4 (100 µM) and lower than that of 3 (100 µM).
The degree of block of the peak currents produced by
10, expressed as the percent of reduction of the KA- or
ATPA-evoked currents (100%), was higher than that
elicited by 1 (Table 4) giving an explanation at receptor
level of the higher capability of 10, in comparison to 1,
to block seizures.
Monocyclic derivatives 10 and 21 will be taken by us
as lead compounds for further investigations of new
AMPA/KA antagonists which could find applications in
the treatment of acute or chronic neurodegenerative
diseases.
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 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-
1
trometer. H NMR spectra of compounds 10 and 29 were also
recorded in acetone-d6 and DMSO-d6. Chemical shifts are
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. NOESY spectra were
carried out by using the standard software package. LC-MS
experiments were performed on a Finnigan MAT LCQ ion trap
mass spectrometer equipped with an APCI interface (source
current 5 mA; capillary temperature 150 °C; vaporizer tem-
perature 600 °C) and connected to a Waters 616 chromato-
graph equipped with a Waters 996 photodiode array detector.
The mass spectrometer operated in a positive-ion mode, with
a scan range from m:z 150 to 800. The HPLC separation was
done by reverse-phase chromatography with a LC-18 column
(250 × 4.6 mm, 5 µm): eluant 0.05% TFA in water/CH3CN
68:32, flow rate 200 µL/min, λ 254 nm.
Because derivatives 10 and 21 reduced KA-evoked
currents, their neuroprotective properties toward a KA-
induced toxicity in primary cultures of cortical neurons
were tested. The application of KA (100 µM) to eight
DIV (days in vitro) neurons for 3 h induced a 70% cell
death. The co-application of KA and derivative 10 or
21 (100 µM) reduced significantly neuronal cell death
to 45% and 55%, respectively (Figure 2). Nevertheless,
compounds 10 and 21 did not completely abolish neu-
ronal cell death induced by KA.
Gen er a l P r oced u r e for th e Syn th esis of 3-N-Alk ylca r -
bam oyl-3,5-dih ydr o-7,8-m eth ylen edioxy-1-(4-n itr oph en yl)-
4H-2,3-ben zod ia zep in -4-on es (25-28). To a solution of 24
(0.5 g, 1.54 mmol) in CH2Cl2 (60 mL) were added triethylamine
(2 mL, 14.4 mmol) and the suitable isocyanate (7.7 mmol). The
reaction mixture was stirred at room temperature for 36 h and
then concentrated in vacuo. The resulting residue was purified
by silica gel column chromatography with CHCl3/EtOAc (70:
30) as eluant and recrystallized from EtOAc to give 25-28.
3,5-Dih ydr o-3-N-m eth ylcar bam oyl-7,8-m eth ylen edioxy-
1-(4-n itr op h en yl)-4H-2,3-ben zod ia zep in -4-on e (25). Mp
194-196 °C (530 mg, 90%). 1H NMR 2.94 (d, 3H, J ) 4.7, CH3),
3.54 (m, 2H, CH2-5), 6.08 (m, 2H, OCH2O), 6.59 (s, 1H, H-9),
6.91 (s, 1H, H-6), 7.95 (d, 2H, J ) 8.9, H-2′,6′), 8.29 (d, 2H, J
) 8.9, H-3′,5′), 8.63 (bs, 1H, NH). Anal. (C17H14N4O6) C, H, N.
In conclusion, novel 2-[(4-alkylsemicarbazono)-(4-ami-
nophenyl)-methyl]-4,5-methylenedioxyphenylacetic acid
alkyl esters reported in this study possess a remarkable
anticonvulsant activity and exhibit lower toxicity than
that shown by compound 1. In particular, derivative 10
is 5-fold more potent than GYKI 52466 and is slightly
more active than its structurally related bicyclic deriva-
tive 4. As a consequence, the data reported in this paper
indicate that the 2,3-diazepine nucleus is not an es-
sential structural requirement for anticonvulsant activ-
ity and can be replaced by a side chain carrying a moiety
capable to mimic the heterocyclic ring.

Novel Potent AMPA/ Kainate Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4439
3,5-Dih yd r o-3-N-eth ylca r ba m oyl-7,8-m eth ylen ed ioxy-
1-(4-n itr op h en yl)-4H-2,3-ben zod ia zep in -4-on e (26). Mp
176-178 °C (537 mg, 88%). 1H NMR 1.22 (t, 3H, J ) 7.2, CH3),
3.39 (m, 2H, NHCH2), 3.54 (m, 2H, CH2-5), 6.08 (m, 2H,
OCH2O), 6.59 (s, 1H, H-9), 6.91 (s, 1H, H-6), 7.95 (d, 2H, J )
8.8, H-2′,6′), 8.28 (d, 2H, J ) 8.8, H-3′,5′), 8.70 (bs, 1H, NH).
Anal. (C18H16N4O6) C, H, N.
(14). With a similar procedure, 14 was prepared from 26 and
anhydrous MeOH. Mp 186-189 °C (382 mg, 80%). 1H NMR
1.24 (t, 3H, J ) 7.1, CH3), 3.30 (m, 2H, CH2), 3.39 (m, 2H,
NHCH2CH3), 3.52 (s, 3H, OCH3), 3.82 (bs, 2H, NH2), 6.05 (m,
2H, OCH2O), 6.23 (m, 1H, NHCH2), 6.56 (s, 1H, H-3), 6.61 (d,
2H, J ) 8.8, H-3′,5′), 6.90 (s, 1H, H-6), 7.29 (d, 2H, J ) 8.8,
H-2′,6′), 7.33 (bs, 1H, NH). Anal. (C20H22N4O5) C, H, N.
3,5-Dih yd r o-7,8-m et h ylen ed ioxy-1-(4-n it r op h en yl)-3-
N-p r op ylca r ba m oyl-4H-2,3-ben zod ia zep in -4-on e (27). Mp
155-157 °C (442 mg, 70%). 1H NMR 0.92 (t, 3H, J ) 7.2, CH3),
1.58 (m, 2H, CH2CH3), 3.32 (m, 2H, NHCH2), 3.54 (m, 2H, CH2-
5), 6.08 (m, 2H, OCH2O), 6.59 (s, 1H, H-9), 6.91 (s, 1H, H-6),
7.95 (d, 2H, J ) 8.8, H-2′,6′), 8.29 (d, 2H, J ) 8.8, H-3′,5′),
8.75 (bs, 1H, NH). Anal. (C19H18N4O6) C, H, N.
3-N-Bu tylca r ba m oyl-3,5-d ih yd r o-7,8-m eth ylen ed ioxy-
1-(4-n itr op h en yl)-4H-2,3-ben zod ia zep in -4-on e (28). Mp
153-156 °C (438 mg, 67%). 1H NMR 0.93 (t, 3H, J ) 7.2, CH3),
1.35 (m, 2H, CH2CH3), 1.58 (m, 2H, NHCH2CH2), 3.36 (m, 2H,
NHCH2), 3.54 (m, 2H, CH2-5), 6.08 (m, 2H, OCH2O), 6.59 (s,
1H, H-9), 6.91 (s, 1H, H-6), 7.95 (d, 2H, J ) 9.0, H-2′,6′), 8.28
(d, 2H, J ) 9.0, H-3′,5′), 8.73 (bs, 1H, NH). Anal. (C20H20N4O6)
C, H, N.
(Z)-2-[(4-Am in oph en yl)-(4-m eth ylsem icar bazon o)-m eth -
yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid Meth yl Ester
(10). To a stirred solution of 3-N-methylcarbamoyl-3,5-dihydro-
7,8-methylenedioxy-1-(4-nitrophenyl)-4H-2,3-benzodiazepin-4-
one (25) (1.2 mmol) in anhydrous MeOH (50 mL), tin(II)-
chloride (6 mmol) was added. The reaction mixture was heated
at 80 °C for 2 h, and before cooling it was filtered over activated
carbon. The solvent was removed under vacuum, and the
resulting residue was dissolved in EtOAc and neutralized with
2 N NaOH (2 × 100 mL). The organic layer was dried (Na2-
SO4) and the solvent was removed at reduced pressure. The
residue was purified by silica gel column chromatography
using EtOAc/cyclohexane/i-PrOH (60:30:10) as eluant and
recrystallized from EtOAc to give 10 (378 mg, 82%). Rf ) 0.55;
mp 209-212 °C; HPLC 5.03 min; MS 385[M + 1], 353, 328,
311, 296. 1H NMR 2.94 (d, 3H, J ) 4.9, NHCH3), 3.30 (m, 2H,
CH2), 3.52 (s, 3H, OCH3), 3.83 (bs, 2H, NH2), 6.05 (m, 2H,
OCH2O), 6.41 (m, 1H, NHCH3), 6.56 (s, 1H, H-3), 6.60 (d, 2H,
J ) 8.7, H-3′,5′), 6.90 (s, 1H, H-6), 7.29 (d, 2H, J ) 8.7, H-2′,6′),
7.39 (bs, 1H, NH). Anal. (C19H20N4O5) C, H, N.
(Z)-2-[(4-Am in op h en yl)-(4-eth ylsem ica r ba zon o)-m eth -
yl]-4,5-m et h ylen ed ioxyp h en yla cet ic Acid E t h yl E st er
(15). With a similar procedure, 15 was prepared from 26 and
1
anhydrous EtOH. Mp 75-78 °C (386 mg, 78%). H NMR 1.12
(t, 3H, J ) 7.1, OCH2CH3), 1.24 (t, 3H, J ) 7.2, NHCH2CH3),
3.28 (m, 2H, CH2), 3.38 (m, 2H, NHCH2CH3), 3.85 (bs, 2H,
NH2), 3.98 (m, 2H, OCH2CH3), 6.05 (m, 2H, OCH2O), 6.23 (m,
1H, NHCH2), 6.55 (s, 1H, H-3), 6.60 (d, 2H, J ) 8.8, H-3′,5′),
6.92 (s, 1H, H-6), 7.30 (d, 2H, J ) 8.8, H-2′,6′), 7.32 (bs, 1H,
NH). Anal. (C21H24N4O5) C, H, N.
(Z)-2-[(4-Am in op h en yl)-(4-eth ylsem ica r ba zon o)-m eth -
yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid P r op yl Ester
(16). With a similar procedure, 16 was prepared from 26 and
1
anhydrous PrOH. Mp 64-67 °C (297 mg, 58%). H NMR 0.82
(t, 3H, J ) 7.4, O(CH2)2CH3), 1.24 (t, 3H, J ) 7.1, NHCH2CH3),
1.51 (m, 2H, OCH2CH2CH3), 3.29 (m, 2H, CH2), 3.38 (m, 2H,
NHCH2CH3), 3.86 (bs, 2H, NH2), 3.87 (m, 2H, OCH2CH2CH3),
6.04 (m, 2H, OCH2O), 6.24 (m, 1H, NHCH2), 6.55 (s, 1H, H-3),
6.60 (d, 2H, J ) 8.8, H-3′,5′), 6.92 (s, 1H, H-6), 7.29 (d, 2H, J
) 8.8, H-2′,6′), 7.32 (bs, 1H, NH). Anal. (C22H26N4O5) C, H, N.
(Z)-2-[(4-Am in op h en yl)-(4-eth ylsem ica r ba zon o)-m eth -
yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid Bu th yl Ester
(17). With a similar procedure, 17 was prepared from 26 and
anhydrous BuOH. Mp 62-65 °C (286 mg, 54%). 1H NMR 0.87
(t, 3H, J ) 7.4, O(CH2)3CH3), 1.24 (t, 3H, J ) 7.1, NHCH2CH3),
1.25 (m, 2H, OCH2CH2CH2CH3), 1.47 (m, 2H, OCH2CH2CH2-
CH3), 3.29 (m, 2H, CH2), 3.39 (m, 2H, NHCH2CH3), 3.85 (bs,
2H, NH2), 3.92 (m, 2H, OCH2CH2CH2CH3), 6.04 (m, 2H,
OCH2O), 6.23 (m, 1H, NHCH2), 6.55 (s, 1H, H-3), 6.60 (d, 2H,
J ) 8.8, H-3′,5′), 6.92 (s, 1H, H-6), 7.30 (d, 2H, J ) 8.8, H-2′,6′),
7.32 (bs, 1H, NH). Anal. (C23H28N4O5) C, H, N.
(Z)-2-[(4-Am in oph en yl)-(4-pr opylsem icar bazon o)-m eth -
yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid Meth yl Ester
(18). With a similar procedure, 18 was prepared from 27 and
anhydrous MeOH. Mp 74-77 °C (257 mg, 52%). 1H NMR 0.99
(t, 3H, J ) 7.4, CH3), 1.64 (m, 2H, NHCH2CH2CH3), 3.30 (m,
4H, CH2 and NHCH2CH2CH3), 3.52 (s, 3H, OCH3), 3.82 (bs,
2H, NH2), 6.04 (m, 2H, OCH2O), 6.26 (m, 1H, NHCH2), 6.56
(s, 1H, H-3), 6.60 (d, 2H, J ) 8.8, H-3′,5′), 6.90 (s, 1H, H-6),
7.28 (d, 2H, J ) 8.8, H-2′,6′), 7.33 (bs, 1H, NH). Anal.
(C21H24N4O5) C, H, N.
(Z)-2-[(4-Am in op h en yl)-(4-bu tylsem ica r ba zon o)-m eth -
yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid Meth yl Ester
(19). With a similar procedure, 19 was prepared from 28 and
anhydrous MeOH. Mp 66-69 °C (256 mg, 50%). 1H NMR 0.97
(t, 3H, J ) 7.4, CH3), 1.42 (m, 2H, NHCH2CH2CH2CH3), 1.59
(m, 2H, NHCH2CH2CH2CH3), 3.25-3.38 (m, 4H, CH2 and
NHCH2), 3.52 (s, 3H, OCH3), 3.82 (bs, 2H, NH2), 6.05 (m, 2H,
OCH2O), 6.25 (m, 1H, NHCH2), 6.56 (s, 1H, H-3), 6.61 (d, 2H,
J ) 8.5, H-3′,5′), 6.90 (s, 1H, H-6), 7.28 (d, 2H, J ) 8.5, H-2′,6′),
7.32 (bs, 1H, NH). Anal. (C22H26N4O5) C, H, N.
(Z)-2-[(4-Am in oph en yl)-(4-m eth ylsem icar bazon o)-m eth -
yl]-4,5-m eth ylen ed ioxy-p h en yla cetic Acid Eth yl Ester
(11). With a similar procedure, 11 was prepared from 25 and
anhydrous EtOH. Mp 148-151 °C (382 mg, 80%). 1H NMR
1.11 (t, 3H, J ) 7.1, OCH2CH3), 2.93 (d, 3H, J ) 4.9, NHCH3),
3.28 (m, 2H, CH2), 3.85 (bs, 2H, NH2), 3.97 (m, 2H, OCH2CH3),
6.04 (m, 2H, OCH2O), 6.23 (m, 1H, NHCH3), 6.54 (s, 1H, H-3),
6.59 (d, 2H, J ) 8.5, H-3′,5′), 6.91 (s, 1H, H-6), 7.28 (d, 2H, J
) 8.5, H-2′,6′), 7.42 (bs, 1H, NH). Anal. (C20H22N4O5) C, H, N.
(Z)-2-[(4-Am in op h e n yl)-(4-m e t h ylse m ica r b a zon o)-
m eth yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid P r op yl
Ester (12). With a similar procedure, 12 was prepared from
1
25 and anhydrous PrOH. Mp 108-111 °C (381 mg, 77%). H
NMR 0.82 (t, 3H, J ) 7.4, O(CH2)2CH3), 1.51 (m, 2H,
OCH2CH2CH3), 2.93 (d, 3H, J ) 4.9, NHCH3), 3.29 (m, 2H,
CH2), 3.85 (bs, 2H, NH2), 3.89 (m, 2H, OCH2CH2CH3), 6.04
(m, 2H, OCH2O), 6.21 (m, 1H, NHCH3), 6.54 (s, 1H, H-3), 6.59
(d, 2H, J ) 8.5, H-3′,5′), 6.92 (s, 1H, H-6), 7.29 (d, 2H, J ) 8.5,
H-2′,6′), 7.36 (bs, 1H, NH). Anal. (C21H24N4O5) C, H, N.
(Z)-2-[(4-Am in op h e n yl)-(4-m e t h ylse m ica r b a zon o)-
m eth yl]-4,5-m eth ylen ed ioxyp h en yl a cetic Acid Bu tyl
Ester (13). With a similar procedure, 13 was prepared from
25 and anhydrous BuOH. Mp 77-80 °C (282 mg, 55%). 1H
NMR 0.87 (t, 3H, J ) 7.1, O(CH2)3CH3), 1.25 (m, 2H, OCH2-
CH2CH2CH3), 1.47 (m, 2H, OCH2CH2CH2CH3), 2.94 (d, 3H, J
) 4.9, NHCH3), 3.29 (m, 2H, CH2), 3.84 (bs, 2H, NH2), 3.92
(m, 2H, OCH2CH2CH2CH3), 6.05 (m, 2H, OCH2O), 6.21 (m, 1H,
NHCH3), 6.55 (s, 1H, H-3), 6.59 (d, 2H, J ) 8.5, H-3′,5′), 6.92
(s, 1H, H-6), 7.29 (d, 2H, J ) 8.5, H-2′,6′), 7.35 (bs, 1H, NH).
Anal. (C22H26N4O5) C, H, N.
(E )-2-[(4-Am in op h e n yl)-(4-m e t h ylse m ica r b a zon o)-
m eth yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid Meth yl
Ester (29). A CHCl3 solution (25 mL) of compound 10 (100
mg) left aside for two week at room temperature yielded a 70:
30 mixture of 10 and 29. Compound 29 was obtained in a pure
form by a silica gel column chromatography using EtOAc/
cyclohexane/i-PrOH (60:30:10) as eluant and was then crystal-
lized from EtOAc. Rf ) 0.42; mp 207-210 °C; HPLC 7.25 min.
1
MS 385[M + 1], 353, 328, 311, 296. H NMR 2.90 (d, 3H, J )
4.7, NHCH3), 3.68 (s, 3H, OCH3), 3.77 (m, 2H, CH2), 3.92 (bs,
2H, NH2), 5.94 (m, 2H, OCH2O), 6.53 (s, 1H, H-3), 6.54 (m,
1H, NHCH3), 6.71 (d, 2H, J ) 8.5, H-3′,5′), 6.79 (s, 1H, H-6),
7.02 (d, 2H, J ) 8.5, H-2′,6′), 7.90 (bs, 1H, NH). Anal.
(C19H20N4O5) C, H, N.
(Z)-2-[(4-Am in op h en yl)-(4-eth ylsem ica r ba zon o)-m eth -
yl]-4,5-m eth ylen ed ioxyp h en yla cetic Acid Meth yl Ester
2-(4-Nitr oben zyl)-4,5-m eth ylen edioxyph en ylacetic Acid
Meth yl Ester (31). To a solution of 2-(4-nitrobenzoyl)-4,5-

4440 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Micale et al.
methylenedioxyphenylacetic acid methyl ester (30) (1.0 g, 2.9
mmol) in anhydrous THF (40 mL), were added BF3OEt2 (1.1
mL, 8.7 mmol) and sodium cyanoborohydride (364 mg, 5.8
mmol). The reaction mixture was refluxed overnight, cooled
at room temperature, and diluted with diethyl ether (40 mL).
The organic solution was sequentially treated with a saturated
NaHCO3 solution and brine, and then dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure, and
the residue was purified by column chromatography using
light petroleum/diethyl ether (70:30) as eluant to give 0.8 g
(83%) of 31 as a white solid. Mp 119-122 °C. 1H NMR 3.47 (s,
2H, CH2COOCH3), 3.60 (s, 3H, CH3), 4.04 (s, 2H, PhCH2), 5.96
(s, 2H, OCH2O), 6.59 and 6.76 (2s, 2H, H-3 and H-6), 7.26 (d,
2H, J ) 8.5, H-2′,6′), 8.13 (d, 2H, J ) 8.5, H-3′,5′). Anal.
(C17H15NO6) C, H, N.
1-[2-(4-Am in ob e n zoyl)-4,5-m e t h yle n e d ioxyp h e n yl-
a cetyl]-3-m eth ylu r ea (22). To a solution of 33 (354 mg, 0.95
mmol) in acetone (100 mL), potassium permanganate (1.42 mg)
and anhydrous sodium sulfate (708 mg) were added, and the
mixture was stirred at room temperature until the disappear-
ance of the starting material (TLC monitoring). Excess of
potassium permanganate was decomposed by adding EtOH.
The reaction mixture was filtered off on a Celite pad and the
filtrate was evaporated. The crude product was purified by
column chromatography with CCl4/EtOAc/i-PrOH as eluant
to give 220 mg (60%). The subsequent treatment with am-
monium formate and Raney-Ni as reported for compound 21,
gave a crude product that was purified by column chromatog-
raphy using EtOAc/cyclohexane/i-PrOH, 60:30:10 as eluant to
give 162 mg (80%) of 22. Mp 109-112 °C. 1H NMR 2.85 (d,
3H, J ) 4.7, CH3), 3.55 (s, 2H, CH2CO), 4.38 (bs, 2H, NH2),
6.04 (s, 2H, OCH2O), 6.65 (d, 2H, J ) 8.5, H-3′,5′), 6.89 (s, 1H,
H-3), 6.92 (s, 1H, H-6), 7.70 (d, 2H, J ) 8.5, H-2′,6′), 8.15 (bs,
1H, NHCH3), 10.1 (bs, 1H, NH). Anal. (C18H17N3O5) C, H, N.
1-(3,4-Meth ylen edioxyph en ylacetyl)-3-m eth ylu r ea (23).
3,4-Methylenedioxyphenylacetic acid (34) (500 mg, 2.78 mmol)
was refluxed with excess of thionyl chloride (10 mL) for 6 h.
Excess of SOCl2 was evaporated under reduced pressure, and
the residue was dissolved in dry benzene and treated with
methylurea (309 mg, 4.17 mmol). The reaction mixture was
refluxed for 6-7 h, then washed with 2% NaHCO3 and
extracted with EtOAc (2 × 100 mL). The combined extracts
were dried over Na2SO4, and the solvent was removed under
pressure to yield a crude product which, by treatment with
acetone, afforded 492 mg (75%) of 23. Mp 162-165 °C. 1H NMR
2.87 (d, 3H, J ) 4.7, CH3), 3.56 (s, 2H, CH2), 5.96 (s, 2H,
OCH2O), 6.75-6.80 (m, 3H, Ar), 8.33 (bs, 1H, NHCH3), 9.30
(bs, 1H, NH). Anal. (C11H12N2O4) 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
KC 18F 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 3:1 (v/v) mixture of acetone and water.
Each compound was dissolved in CHCl3 (2 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 are expressed as the mean values
of five determinations. The Rm values were calculated from
the experimental Rf values according to the equation Rm ) log-
[(1/Rf) - 1]. Higher Rm values indicate higher lipophilicity. The
log P values reported in Table 1 were calculated by using the
XLOGP v 2.0 program.18 Calculated log P and experimental
Rm values are linearly correlated with an r2 value of 0.86 at
95% confidence limits.
Mod elin g Stu d ies. Calculations on compounds 10 and 29
were performed with the PC Spartan Pro molecular modeling
program (Wavefunction, Inc., Irvine, CA) using the semiem-
pirical AM1 method13 and were carried out at the RHF level.
The molecules were built from the model kit containing the
atomic fragments and were subjected to conformational search
through the conformational distribution option. Both com-
pounds showed several minimum energy conformations of
comparable energy. The energy barriers for rotation around
the single bond connecting the semicarbazono moiety to C-2
were determined through location of the transition states
corresponding to the maxima of the energy profiles.
2-(4-Am in ob en zyl)-4,5-m et h ylen ed ioxyp h en yla cet ic
Acid Meth yl Ester (20). A suspension of 31 (400 mg, 1.2
mmol) and Raney-Ni (60 mg) in EtOH (20 mL) was stirred
with ammonium formate (120 mg) at room temperature. After
completion of the reaction (monitored by TLC EtOAc/cyclo-
hexane, 1:1), the mixture was filtered off. The organic layer
was evaporated under reduced pressure, and the residue
dissolved in CHCl3 was washed with saturated NaCl to remove
ammonium formate. The organic layer was evaporated under
reduced pressure and the residue was purified by treatment
1
with acetone to give 294 mg, 81% of 20. Mp 150-153 °C. H
NMR 3.53 (s, 2H, CH2CO), 3.64 (s, 3H, CH3), 3.82 (s, 2H,
PhCH2), 3.86 (bs, 2H, NH2), 5.92 (s, 2H, OCH2O), 6.62 and
6.73 (2s, 2H, H-3 and H-6), 6.61 (d, 2H, J ) 8.0, H-3′,5′), 6.89
(d, 2H, J ) 8.0, H-2′,6′). Anal. (C17H17NO4) C, H, N.
2-(4-Nitr oben zyl)-4,5-m eth ylen edioxyph en ylacetic Acid
(32). To a suspension of 31 (800 mg, 2.4 mmol) in H2O (60
mL) were added THF (15 mL) and HCl 6 N (4 mL). The
mixture was refluxed for 9 h, cooled at room temperature, and
then diluted with H2O (60 mL) and extracted with EtOAc (2
× 60 mL). The combined extracts were evaporated under
reduced pressure, and the residue was purified by treatment
with diethyl ether to give 500 mg, 65% of 32. Mp 222-224 °C.
1H NMR 3.51 (s, 2H, CH2COOH), 4.04 (s, 2H, PhCH2), 5.97
(s, 2H, OCH2O), 6.60 and 6.77 (2s, 2H, H-3 and H-6), 7.26 (d,
2H, J ) 8.5, H-2′,6′), 8.13 (d, 2H, J ) 8.5, H-3′,5′). Anal.
(C16H13NO6) C, H, N.
1-[2-(4-Nitr oben zyl)-4,5-m eth ylen ed ioxyp h en yla cetyl]-
3-m eth ylu r ea (33). A mixture of compound 32 (500 mg, 1.6
mmol) and excess of thionyl chloride (8 mL) was heated at
reflux for 6 h. Excess of thionyl chloride was removed under
reduced pressure, and the residue was dissolved in benzene
(50 mL) and treated with methylurea (176 mg, 2.4 mmol) at
reflux (6 h). The resulting mixture was poured into saturated
aqueous NaHCO3 and extracted with EtOAc (2 × 50 mL). The
combined extracts were dried over Na2SO4 and purified by
column chromatography with CCl4/EtOAc/i-PrOH (70:20:10)
as eluant to give 354 mg (60%) of 33. Mp 207-210 °C. 1H NMR
2.79 (d, 3H, J ) 4.7, CH3), 3.50 (s, 2H, CH2CO), 4.02 (s, 2H,
PhCH2), 6.00 (s, 2H, OCH2O), 6.67 (s, 1H, H-3), 6.76 (s, 1H,
H-6), 7.24 (d, 2H, J ) 8.5, H-2′,6′), 8.12 (d, 2H, J ) 8.5, H-3′,5′),
8.14 (bs, 1H, NHCH3), 8.26 (bs, 1H, NH). Anal. (C18H17N3O6)
C, H, N.
1-[2-(4-Am in oben zyl)-4,5-m eth ylen edioxyph en ylacetyl]-
3-m eth ylu r ea (21). To a solution of 33 (354 mg, 0.95 mmol)
in EtOH (30 mL) was added ammonium formate (96 mg, 1.5
mmol) and Raney-Ni (50 mg), and the mixture was heated
at reflux for 2 h. The mixture was filtered off on a Celite pad
and the solvent was removed under reduced pressure. The
residue, dissolved in CHCl3, was washed with saturated NaCl
to remove ammonium formate. The organic layer, after
evaporation of the solvent, gave the desired amino derivative
21 which was purified by treatment with acetone (273 mg,
84%). Mp 199-202 °C. 1H NMR 2.82 (d, 3H, J ) 4.7, CH3),
3.51 (s, 2H, CH2CO), 3.59 (bs, 2H, NH2), 3.76 (s, 2H, PhCH2),
5.87 (s, 2H, OCH2O), 6.61 (d, 2H, J ) 8.2, H-3′,5′), 6.67 (s, 1H,
H-3), 6.71 (s, 1H, H-6), 6.86 (d, 2H, J ) 8.2, H-2′,6′), 7.16 (bs,
1H, NH), 8.12 (bs, 1H, NHCH3). Anal. (C18H19N3O4) C, H, N.
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.19 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 50%
DMSO and 50% sterile saline (0.9% NaCl). Individual mice
were placed under a hemispheric Perspex dome (diameter 58

Novel Potent AMPA/ Kainate Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4441
cm) and were allowed 60 s for habituation. Assessment of
locomotor activity was also made during this time interval.
Auditory stimulation (12-16 kHz, 109 dB) was applied for 60
s or until tonic extension occurred and induced a sequential
seizure response in control DBA/2 mice, consisting of an early
wild running phase, followed by generalized myoclonus and
tonic flexion and extension, sometimes followed by respiratory
arrest. The control and drug-treated mice were scored for
latency to and incidence of the different phases of the audio-
genic seizures.20 The time course of the anticonvulsant action
of 10 was determined following the administration of 33 µmol/
kg of compound to groups of 10 mice for each time. The animals
were tested for sound-induced seizure responses from 15 to
120 min after drug administration.
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
et al.21 Abolition of tonic hindlimb extension after drug
treatment was considered as the endpoint of protection. The
dose-response curves were estimated by testing four to five
doses using 8-10 mice for each dose.
noethyl ether)N,N,N′,N′-tetraacetic acid (EGTA) 5, N-(2-hy-
droxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) 5,
ATP-Na 2, pH 7.3 with KOH. The cells were continuously
perfused with the external solution (mM): NaCl 145, KCl 5,
CaCl2 1, HEPES 5, glucose 5, sucrose 20, pH 7.4 with NaOH.
All drugs were dissolved in DMSO and diluted at the final
concentration (<1%) in extracellular solution. KA was also
dissolved in the extracellular solution. All drugs were applied
directly by gravity through a Y-tube perfusion system26 at
same concentration (100 µM). After a wash-out of compounds
10 and 21 from the cell culture, the responses to KA returned
to the control level.
Effects on Motor Movem en ts. Male Swiss mice (20-26
g, 48-54 days old) were purchased from Charles River. Groups
of 10 mice were trained to do coordinated motor movements
continuously for 2 min on a rotarod, 3 cm in diameter, at 8
rpm (U. Basile, Comerio, Varese, Italy). Impairment of coor-
dinated motor movements was defined as the inability of the
mice to remain on the rotarod for a 2-min test period.27 The
ability of the mice to remain on the rotarod was tested 30 min
after administration of various compounds.
Excitotoxic Exp er im en ts.28 Drugs were dissolved in Mg-
free Loke’s buffer (NaCl 154 mM, KCl 5.6 mM, CaCl2 2.3 m,
NaHCO3 3.6 mM, D-glucose 5.5 mM, Hepes 5 mM, pH 7.4).
The medium was removed from the cell culture and a solution
of the test drug (KA 100 µM without or with 100 µM 10 or 21)
was added. The cultures were incubated for 3 h at 37 °C. The
experiment was stopped by washing the cultures with the
Loke’s Buffer later replaced with the culture medium. Neu-
ronal cell death was assessed 24 h later and was estimated
by the MTT tetrazolium salt assay.28 The adsorbance were
recorded at 570 nm and 630 nm.
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 the sc administration of pentylenetetrazole (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 50% DMSO and 50% sterile saline (0.9% NaCl).
The convulsive dose 97 (CD97) of PTZ (85 mg/kg) was applied,
and the animals were observed for 30 min. A threshold
convulsion was an episode of clonic spasms lasting for at least
5 s. The absence of this threshold convulsion over 30 min
indicated that the tested substance had the ability to elevate
PTZ seizure threshold.22
AMP A-In d u ced Seizu r es in DBA/2 Mice. The CD50 ((
95% confidence limits) of icv microinjected AMPA was 1.76
(1.06-3.07) nmol for clonus and 2.90 (1.83-4.58) nmol for
tonus. For icv injection, the 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;23 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.
Sta tistica l An a lysis. The ED50 values of each phase of the
audiogenic seizure or seizures induced by MES, PTZ, AMPA,
or KA were determined for each compound administered, and
dose-response curves were fitted using a computer program
by the method of Litchfield and Wilcoxon.29 The relative
anticonvulsant activities were determined by comparison of
respective ED50 values. The median toxic dose (TD50) values
were estimated using the method of Litchfield and Wilcoxon.29
The relative activities were determined by comparison of
respective TD50 values. Statistical significance between control
and test groups of data means was tested using a two-tailed
Student’s t test. Electrophysiological data were analyzed using
the software Clampex (Axon Instrument). Results are ex-
pressed as mean ( SE. Origin (Microcal Software, Northamp-
ton, MA) was used for figure preparation and statistical
analysis.
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 compound 10. Animals
showing 5 s or more of clonic activity were scored as not
protected according to Donevan et al.6 The period of observa-
tion was 60 min.
Ack n ow led gm en t. This work was financially sup-
ported by the Ministero dell’Istruzione, dell’Universita`
e della Ricerca (MIUR-COFIN2001), Rome, Italy.
Electr op h ysiology. Primary cultures of cerebellar granule
neurons were prepared from 7 to 8 days old Sprague-Dawley
rats as previously described.24 Cells from cerebella were
dispersed with trypsin (0.24 mg/mL) and plated at a density
of 106 cells/mL on 35-mm Falcon dishes coated with poly-L-
lysine (10 µg/mL). The cells were grown in basal Eagle’s
medium, supplemented with 10% fetal bovine serum, 2 mM
glutamine, and 100 µg/mL gentamycin, and maintained at 37
°C in 5% CO2. Cytosine arabinofuranoside (10 µM) was added
to the cultures 24 h after plating to prevent astroglia prolifera-
tion.
Electr op h ysiologica l Recor d in gs. Recordings were per-
formed on single cerebellar granule neurons after 7 days in
culture24 using the voltage-clamp technique in the whole-cell
configuration.25 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-
tained (mM): KCl 140, MgCl2 3, ethylene glycol-bis-(â-ami-
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