Structure-activity study of 2,3-benzodiazepin-4-ones noncompetitive AMPAR antagonists: Identification of the 1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one as neuroprotective agent

Article abstract of DOI:10.1016/j.bmc.2007.11.080

In the search for AMPA receptor (AMPAR) antagonists, 2,3-benzodiazepines represent a family of specific noncompetitive antagonists with anticonvulsant and neuroprotective properties. We have previously shown that 2,3-benzodiazepin-4-ones possess marked an

Full text of DOI:10.1016/j.bmc.2007.11.080

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2200–2211
Structure–activity study of 2,3-benzodiazepin-4-ones
noncompetitive AMPAR antagonists: Identification of the
1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-
4H-2,3-benzodiazepin-4-one as neuroprotective agent
a
a
Nicola Micale,a, ,ꢀ Simona Colleoni,b,c,ꢀ Giovanna Postorino, Alessia Pellicano,
*
`
a
d
c
c
`
Maria Zappala, John Lazzaro, Valentina Diana, Alfredo Cagnotto,
Tiziana Menninic and Silvana Grassoa
a
`
Dipartimento Farmaco-Chimico, Universita di Messina, Viale Annunziata, 98168 Messina, Messina, Italy
b
`
Istituto di Chimica Farmaceutica, Universita di Milano, Italy
cIstituto di Ricerche Farmacologiche ‘Mario Negri’, Milano, Italy
dPfizer Global Research and Development, Groton, CT, USA
Received 20 July 2007; revised 22 November 2007; accepted 30 November 2007
Available online 5 December 2007
Abstract—In the search for AMPA receptor (AMPAR) antagonists, 2,3-benzodiazepines represent a family of specific noncompet-
itive antagonists with anticonvulsant and neuroprotective properties. We have previously shown that 2,3-benzodiazepin-4-ones pos-
sess marked anticonvulsant properties and high affinity for the noncompetitive binding site of the AMPAR complex. In this paper,
we report the synthesis and pharmacological characterization of a full set of 2,3-benzodiazepin-4-ones in order to better define the
structure–activity relationship (SAR) of this class of compounds. Binding assays and functional tests were performed to evaluate the
antagonistic activity at the AMPARs. Through these results we have identified a potent AMPAR antagonist, 1-(4-amino-3-meth-
ylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one (5c). This compound noncompetitively inhibited AMPAR-
mediated toxicity in primary mouse hippocampal cultures with an IC50 of 1.6 lM and blocked kainate-induced calcium influx in
rat cerebellar granule cells with an IC50 of 6.4 lM. Thus, 5c has the in vitro potential as therapeutic drug in the treatment of various
neurological disorders.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
of AMPARs as potential targets for therapeutic inter-
vention in many neurological disorders, and the devel-
opment of selective competitive and noncompetitive
antagonists, is one of the main goals in
neuropharmacology.
a-Amino-3-hydroxy-5-methyl-4-isoxazole
propionate
(AMPA) receptors (AMPARs) are glutamate-gated ion
channels which mediate the majority of fast excitatory
synaptic transmissions in the mammalian brain. They
belong to a larger family of ionotropic glutamate recep-
tors including the closely related kainate (KA) and N-
methyl-D-aspartic acid (NMDA) receptors.1 Hyperacti-
vation of AMPARs is implicated in a broad range of
acute and chronic neurodegenerative and neuropsychi-
atric conditions, for example, cerebral ischemia, epi-
lepsy, and Alzheimer’s disease.2 Thus, the modulation
Among the competitive agents, 2,3-dihydroxy-6-nitro-
7-sulfamoyl-benzo[f]quinoxaline (NBQX) has been
shown to be antiepileptic and neuroprotective in a vari-
ety of tests of cerebral ischemia and neurodegenera-
tion. However, NBQX and its analogues have been
recently shown to increase c-amino-butyric acid
(GABA) transmission in the cerebellum by non-
AMPA-dependent mechanism, to depolarize hippo-
campal, and also to act at the KA receptors,
implying that these agents may not be selective.3 Non-
competitive AMPAR antagonists have the theoretical
advantage to counteract excitotoxicity even at high
Keywords: 2,3-Benzodiazepin-4-ones; AMPAR affinity; Binding assay.
*
Corresponding author. Tel.: +39 090 6766466; fax: +39 090
355613; e-mail: nmicale@pharma.unime.it
Authors contributed equally to this work.
ꢀ
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.080

N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
2201
concentration of glutamate and to show less side-effects
than competitive antagonists.4 To date, there are a
number of pharmacological agents that affect AMPAR
function through interactions outside of the agonist-
binding domain.5 Among them there are 2,3-benzodi-
azepines (2,3-BZs) noncompetitive AMPAR antago-
nists whose prototype, 1-(4-aminophenyl)-4-methyl-
state of the GluR2Qflip, a key subunit that controls the
calcium permeability of heteromeric AMPARs and thus
is involved in the etiology of neurodegenerative
disorders.11
By introducing a methyl group at C-5 of the diazepine
nucleus, we have recently demonstrated a stereoselective
interaction of 2a (Fig. 1) with the noncompetitive bind-
ing site of the AMPARs and that (5S)-(ꢀ)-2a resulted to
be the eutomer. Noteworthy, the configuration of (5S)-
(ꢀ)-2a is opposite to that of Talampanel. However, a
superimposition of the most populated conformation
of the two molecules reveals an excellent overlapping
of the common functional groups.12 Moreover, in a re-
cent paper we reported preliminary results that showed
an improvement toward AMPAR affinity of 2,3-ben-
zodiazepin-4-ones by substituting the dioxole nucleus
with the dioxane homologue (i.e., 5a, 5c, and 5f).13
7,8-methylenedioxy-5H-2,3-benzodiazepine
(GYKI
52466) (Fig. 1), demonstrated significant anticonvul-
sant and neuroprotective action.6 These antagonists
bind at the interface between the S1 and S2 glutamate
binding core and channel transmembrane domains,
specifically interacting with S1-M1 and S2-M4 linkers,
thereby disrupting the transduction of agonist binding
into channel opening.7
Highly active analogues of GYKI 52466 have been
found, for example, 3,4-dihydro-3-N-methylcarbamoyl
(GYKI 53655) and 3,4-dihydro-3-N-acetyl (GYKI
53405) derivatives (Fig. 1).8 The enantioselectivity of
these derivatives has been evaluated, and the eutomer
turned out to be the (4R)-enantiomer.6 Subsequently,
the eutomer (4R)-(+) of GYKI 53405 (Talampanel)
was chosen as a drug candidate and is now in clinical
investigation as an anticonvulsant and antiischemic.9
All these interesting results prompted us to synthesize a
complete set of 2,3-benzodiazepin-4-ones in which we
conveyed into our lead structures 1a, 2a, and 5a the
most remarkable modifications observed throughout
our previous research efforts with the aim to gain more
information from SAR studies and optimize the phar-
macological profile of this class of noncompetitive AM-
PAR antagonists.
In the course of our studies we synthesized 1-(4-amino-
phenyl)-3,5-dihydro-7,8-methylenedioxy-4H-2,3-ben-
zodiazepin-4-one (1a) and its 3-N-methylcarbamoyl
derivative (1b) (Fig. 1), which have been shown to inter-
act with the same allosteric AMPAR binding site of
GYKI 52466.10 Furthermore, kinetic investigations of
the mechanism of inhibition performed by using a la-
ser-pulse photolysis technique put in evidence that this
class of compounds preferably inhibits the open-channel
To evaluate the AMPAR antagonism, all compounds
have been screened against KA-induced Ca2+ uptake,
which is mediated by AMPARs, in primary cultures of
rat cerebellar granule neurons, and their interaction with
the noncompetitive binding site labeled by [3H]CP-
526,427 has been assessed. Several active compounds
were found, among which 1-(4-amino-3-methylphenyl)-
3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-
one (5c) disclosed the highest potency and so it was se-
lected for a study as neuroprotective agent. Therefore,
we have established an in vitro model of AMPAR-med-
iated excitotoxicity utilizing mouse embrionic hippo-
campal cultures exposed to KA.
CH3
CH3
O
O
R
O
O
O
N
N
N
N
2. Results and discussion
2.1. Chemistry
H2N
H2N
GYKI 53655, R=NHCH3
GYKI 53405, R=CH3
GYKI 52466
R
The synthesis of target compounds was accomplished
according to the reaction sequence reported in Scheme
1. Methyl 3,4-methylenedioxyphenylacetate (6) was con-
verted in high yield into the corresponding a-methyl 712
or a-ethyl derivatives 8 by a-alkylation with the appro-
priate alkyl iodide conducted in THF and in the pres-
ence of potassium hydride. A hindered strong base
such as potassium hexamethyldisilazane (KHMDS)
was necessary to efficiently form the kinetic enolate of
the carbonyl group and to introduce thereby the benzyl
group (9). Intermediate ketoesters 10–15 were easily pre-
pared via acylation of derivatives 6–9 with the appropri-
ate 4-nitrobenzoic acid in the presence of an excess of
phosphorus pentoxide. The treatment of 10–15 with
hydrazine, followed by reduction with Raney-Ni/ammo-
nium formate, gave 1-(4-aminophenyl)-3,5-dihydro-7,8-
O
O
O
O
NR'
NR'
O
N
N
O
R"
H2N
1a, R=R'=H
1b, R=H, R'=CONHCH3
2a, R=CH3, R'=H
H2N
5a, R'=R"=H
5b, R'=CONHCH3, R" = H
5c, R'=H, R" = CH3
5f, R'=H, R" = Cl
Figure 1.

2202
N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
R
R
O
R
O
b
O
O
c, d
COOCH3
O
O
O
NH
COOCH3
O
N
R"
6 R =H
7 R=CH3
8 R=C2H5
9 R = CH2C6H5
R'
R"
R'
H2N
NO2
a
10, R=H, R'=R''=CH3
11, R=R''=H, R'=OCH3
12, R=R'=CH3, R''=H
1d R=H, R'=R"=CH3
1e R=R''=H, R'=OCH3
2c R=R'=CH3, R=H
13, R=C2H5, R'=R''=H
14, R=C2H5, R'=CH3, R''=H
15, R=CH2C6H5, R'=R''=H
3a R=C2H5, R'=R''=H
3c R=C2H5, R'=CH3, R''=H
4a R=CH2C6H5, R'=R''=H
H3C
H3C
O
H3C
O
O
O
O
O
O
f, g
d, e
O
O
NH
N
NCONHCH3
N
NH
N
Cl
2f
2b
16
H2N
H2N
O2N
R
O
R
R
O
O
O
O
b
COOCH3
O
COOCH3
c, d
NH
N
O
O
17 R =H
18 R=CH3
a
R''
R'
R''
R'
NO2
H2N
19 R=H, R'=R''=CH3
20 R=R''=H, R'=OCH3
21 R=CH3, R'=R''=H
22 R=R'=CH3, R''=H
5d R=H, R'=R''=CH3
5e R=R''=H, R'=OCH3
6a R=CH3, R'=R''=H
6c R=R'=CH3, R''=H
6f R=CH3, R'=Cl, R''=H
e
Scheme 1. Reagents and conditions: (a) CH3I or C2H5I and KH, or BnBr and KHMDS, THF anhydrous, ꢀ70 ꢁC, rt, 16 h; (b) ArCOOH, P2O5,
(CH2Cl)2, rt, 16 h; (c) NH2NH2ÆH2O, ethanol or n-butanol, D, 20 h; (d) HCOONH4, Ni-Raney, EtOH, D, 2 h; (e) NCS, DMF, rt, 20 h; (f) CH3NCO,
CH2Cl2, Et3N, rt, 24 h; (g) H2/5% Pd–C, CHCl3, rt, 3 h.
methylenedioxy-4H-2,3-benzodiazepin-4-ones (1d–1e,
2c, 3a, 3c, 4a) in good yields.
2.2. Pharmacology
2.2.1. Radioligand binding assay and structure–activity
relationships. We have examined compounds 1–6 for
their ability to displace [3H]CP-526,427 from the corre-
sponding binding site of the AMPAR complex.
[3H]CP-526,427 binds in a saturable manner to rat fore-
brain membranes with Kd value 10.47 nM and Bmax va-
lue 4.2 pmol/mg of protein. The inhibition of [3H]CP-
526,427 specific binding (3 nM) to rat forebrain mem-
branes was evaluated as previously described.14 In this
assay, CP-465,022 was used as a positive control; its
IC50 values ranged from 20 to 40 nM. Most of the com-
pounds show an ability to inhibit specific binding similar
to that of GYKI 52466 (1b, 1c, 2a, 2c, 2f, 3a, 3c, 5a, 5b,
6a); whereas some of them have a higher affinity (2b, 5c,
5f, 6c) (Table 1).
Compound 16 by reduction with Raney-Ni/ammonium
formate and successive treatment with N-chlorosuccini-
mide (NCS) afforded compound 2f, whereas by treat-
ment with an excess of methyl isocyanate in the
presence of triethylamine yielded the corresponding 3-
N-methylcarbamoyl derivative, which furnished com-
pound 2b by catalytic hydrogenation with 5% Pd/C. Fol-
lowing analogous procedures, 7,8-ethylenedioxy-4H-
2,3-benzodiazepin-4-ones 5d, 5e, 6a, 6c, and 6f were syn-
thesized starting from methyl 3,4-ethylenedioxypheny-
lacetate/propionate (17, 18), via ketoesters 19–22, as
shown in Scheme 1. Physical and spectral data (1H
NMR) of the synthesized compounds are in agreement
with the proposed structures.

N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
2203
A survey of these results put in evidence that the intro-
duction of a methyl or ethyl group at C-5 of the diaze-
pine nucleus enhances the affinity toward the
noncompetitive binding site of AMPARs (e.g., 1a IC50
32 lM vs 2a IC50 12.9 lM and 3a IC50 8.37 lM), sug-
gesting that a hydrophobic substituent at C-5 could be
suitable for a better fit with the binding site. To evaluate
the presence in the 2,3-BZ binding site of a pocket capa-
ble to accommodate groups more sterically hindered, we
synthesized 5-benzyl derivative 4a. The results clearly
indicate that the insertion of a bulkier substituent
strongly decreases the affinity (4a IC50 > 100 lM), sug-
gesting the presence of a hydrophobic pocket of limited
size in the binding site. Considering that both the intro-
duction of a methyl group at C-5 and the ring enlarge-
ment of the dioxole ring to dioxane homologue
brought to an increase in binding affinity (1a IC50
32 lM vs 5a IC50 9.0 lM), we try to improve the inter-
actions between our compounds and the AMPAR bind-
ing site by inserting concomitantly these two
modifications. However, the insertion of a methyl group
at C-5 in the 7,8-ethylenedioxy derivatives 6 does not
seem to affect significantly the binding affinity (e.g., 5a
IC50 9.0 lM vs 6a IC50 6.96 lM).
manner with IC50 values ranging from 1.23 to 31 lM
(Table 1).
Their ability to reduce Ca2+ uptake is similar or better
than that shown by GYKI 52466.
The results of this investigation are roughly in accor-
dance with the data obtained in the binding experi-
ments. It is clear that compounds containing a 7,8-
ethylenedioxy fragment (5–6) possess a higher antago-
nist activity at the AMPAR than the corresponding
7,8-methylenedioxy homologues (1–2). In fact, com-
pounds 5a, 5b, and 6c are more potent than the corre-
sponding derivatives 1a, 1b, and 2c (5a IC50 2.9 lM,
5b IC50 5.4 lM, 6c IC50 1.23 lM vs 1a IC50 12 lM, 1b
IC50 11 lM, 2c IC50 18.8 lM, respectively).
2.2.3. Kainate-induced toxicity. We have also character-
ized the excitotoxicity of KA in primary cultures of
mouse hippocampal neurones from mouse embryos
(E13) at 7 days in vitro (DIV). Exposure of cultured hip-
pocampal cells to various concentrations of KA (5–
500 lM) for 48 h induced concentration-dependent
neurotoxicity, as revealed by the MTT assay. Nonlinear
regression analysis of viability values yielded an EC50 of
17.3 lM (Fig. 2).
When we add a methyl or chlorine substituent into phe-
nyl ring at C-1 close to the amino group, we generally
observe an improvement of affinity both in the dioxole
series (e.g., 1a IC50 32 lM vs 1c IC50 6.3 lM) and in
the dioxane analogues (e.g., 5a IC50 9.0 lM vs 5c IC50
1.84 lM and 5f IC50 2.05 lM). Noteworthy, this struc-
tural modification should also slow down the first path
metabolism (acetylation of the 4-amino group), as re-
ported for a number of 2,3-BZ analogues that have a
longer duration of action.15
Evidence for KA-induced excitotoxicity through
AMPA-preferring receptors is that NBQX and GYKI
52466 completely blocked this toxicity (KA 30 lM)
(Fig. 3) with an IC50 11.9 0.07 lM and
10.8 0.25 lM, respectively. Using this test, we evalu-
ated the neuroprotective effects of compound 5c
(Fig. 3), which possesses the highest affinity for AM-
PAR (IC50 1.84 lM) and a good in vivo anticonvulsant
activity, as previously reported.13 The results presented
in Figure 4A show that 5c inhibited KA-induced toxicity
completely and concentration-dependently. Compound
5c induced, dose-dependently, a shift of the KA re-
sponses (Fig. 4B); higher KA concentrations than those
shown in Figure 4B were not used because of its poor
solubility and nonspecific effects. Noteworthy, com-
pound 5c showed higher neuroprotective activity than
NBQX and GYKI 52466 with an IC50 value of
1.6 0.02 lM.
Owing to the good AMPAR affinity of compounds 1c,
5c, and 5f, we synthesized a number of derivatives 3,5-
dimethyl or 3-methoxy substituted. However, the pres-
ence of two methyl groups decreases the receptor
affinity (e.g., 1d and 5d IC50 > 100 lM) probably for
a steric hindrance that completely shielded amino
function, an essential structural requirement involved
in the binding with the noncompetitive site of AM-
PARs. Similarly the lack of affinity of the 3-methoxy
substituted derivatives (1e and 5e) might be due to ste-
ric effects. Finally, a methylcarbamoyl moiety at N-3
determines an improvement of affinity in the dioxane
series (e.g., 5a IC50 9.0 lM vs 5b IC50 7.78 lM), as
well as within the dioxole series (e.g., 1a IC50 32 lM
vs 1b IC50 12 lM and 2a IC50 12.9 lM vs 2b IC50
4.01 lM).
Previous studies were carried out on the neuroprotec-
tive efficacy of GYKI 52466, LY300164 (Talampanel,
eutomer of GYKI 53405), and LY303070 (eutomer
of GYKI 53655) in an embryonic rat hippocampal
culture model of AMPA receptor-mediated excitotox-
icity.17,18 These compounds attenuated the KA-excito-
toxicity in a dose-dependent manner with LY300164
and LY303070 being 2.25 and 5.4 times more potent
than GYKI 52466, respectively. Although absolute
comparison of the IC50 values cannot be done due
to species differences (the data that we have obtained
came from embryonic mouse hippocampal culture), in
our study 5c is 6.75 times more potent than GYKI
52466. These findings suggest that 5c has neuroprotec-
tive efficacy comparable to LY303070 and higher than
2.2.2. Intracellular calcium influx. Functional antago-
nism was determined by evaluating the ability of com-
pounds 1–6 to inhibit KA-induced increase of the
[Ca2+]i in primary cultures of rat cerebellar granule cells
(CGC) which express AMPARs (Table 1); GYKI 52466
was used as the control.16
With the exception of 1d and 5d, inactive also in the
binding assay, all compounds completely inhibited the
KA-induced Ca2+ uptake in a concentration-dependent
LY300164,
candidate.19
a
promising neuroprotective drug

2204
N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
Table 1. Pharmacological data of compounds 1–6 and GYKI 52466
R
O
O
[ ] n
NR'
O
N
R"'
R''
H2N
Compound
n
R
R0
R00
R000
[3H]CP-526,427 IC50 (lM)
KA-[Ca2+]i IC50 (lM)
1a
1b
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
H
H
CONHCH3
H
H
H
H
32a
12a
H
H
12a
6.3
11a
2.5
1c
1d
H
H
CH3
CH3
OCH3
H
H
H
CH3
H
>100
>100
12.9
4.01
9.62
11.4
8.37
15.6
>100
9.0
>100
31
12a
1e
2a
H
H
CH3
CH3
CH3
CH3
C2H5
C2H5
CH2Ph
H
H
CONHCH3
H
2b
2c
H
H
15.8
18.8
7.0
H
CH3
Cl
H
2f
3a
H
H
H
H
H
17.1
4.34
ND
2.9a
5.4a
6.4
3c
4a
H
CH3
H
H
H
H
5a
5b
H
CONHCH3
H
H
H
H
H
7.78
1.84a
>100
81.1
2.05a
6.96
4.5
5c
5d
H
H
H
H
H
H
H
H
CH3
CH3
OCH3
Cl
H
H
CH3
H
>100
12
2.0
5e
H
5f
6a
H
H
CH3
CH3
CH3
H
H
ND
1.23
18.0
22a
6c
6f
GYKI 52466
CH3
Cl
H
H
21.6
12.6a
H
IC50 are representative values of three different experiments. Variability is less than 10%.
a Data taken from Refs. 12 and 13.
100
50
0
***
100
***
***
50
0
-
NBQX GYKI 52466
5c
-9
-8
-7
-6
-5
-4
-3
log[KA, M]
KA 30 μM
Figure 2. Dose–response of KA toxicity in mouse hippocampal
cultures. Cultures (7 DIV) were treated with KA at the indicated
concentrations and culture viability was assessed 48 h later. Cell
viability was measured by MTT assay. Each point is the mean SEM
of five–six experiments, carried out in duplicate.
Figure 3. Protective effect of AMPAR antagonist on KA-induced
toxicity. Mouse hippocampal neurones were treated with the com-
pounds (10 lM NBQX, 10 lM GYKI 52466, and 3 lM 5c) concom-
itantly with KA addition (30 lM) for 48 h. The data represent
means SEM of two experiments carried out in triplicate. The
statistical analysis was carried out with the one-way ANOVA followed
by Tukey’s test ***p < 0.001 versus KA.
3. Conclusion
In summary, SAR study of the complete set of 2,3-
benzodiazepinen-4-ones reveals compound 5c as the
most potent AMPAR antagonist. 5c significantly
inhibits [3H]CP-526,427 specific binding, strongly
blocks AMPAR-mediated toxicity in primary mouse
hippocampal cell cultures, and completely reduces cal-
cium influx in rat cerebellar granule neurones. Thus,
5c has the in vitro potential as therapeutic drug in
the treatment of various types of neurological
disorders.

N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
2205
and extracted with diethyl ether (2 · 150 mL). The com-
bined organic layers were washed with a saturated solu-
tion of Na2CO3 and dried over Na2SO4. The solvent was
then removed and the residue was purified by a silica gel
column chromatography (cyclohexane/diethyl ether
60:40) to afford compound 8 (2.9 g, 85%) as a colorless
oil. Rf = 0.68 (cyclohexane/diethyl ether 60:40). 1H
100
50
A
NMR (300 MHz, CDCl3),
d ppm 0.87 (t, 3H,
J = 7.4 Hz, CH3CH2), 1.67–2.06 (m, 2H, CH2CH3),
3.37 (t, 1H, J = 7.7 Hz, CH), 3.65 (s, 3H, OCH3), 5.95
(s, 2H, OCH2O), 6.70–6.87 (m, 3H, Ar). Anal. Calcd
for C12H14O4: C, 64.85; H, 6.35. Found: C, 64.71; H,
6.47.
0
-8
-7
-6
-5
log[compound, M]
100
B
4.2.2. Methyl 2-(3,4-methylenedioxyphenyl)-3-phenylpro-
pionate (9). To a stirred solution of 6 (2 g, 10.3 mmol) in
anhydrous THF (50 mL) at ꢀ70 ꢁC were added
KHMDS (14.23 mL, 10.8 mmol) and benzyl bromide
(1.35 mL, 11.33 mmol) in more portions. The solution
was stirred for 5 h increasing gradually the temperature
until room temperature. The solution was quenched
with sat. NH4Cl (5 mL) and the aqueous phase was ex-
tracted with diethyl ether (2 · 150 mL). The combined
organic layers were dried over Na2SO4. The solvent
was then removed and the residue was purified by silica
gel column chromatography (cyclohexane/diethyl ether
70:30) to afford compound 9 (726 mg, 25%) as a color-
50
0
-6
-5
-4
-3
log[KA, M]
Figure 4. Effect of 5c on KA toxicity in mouse hippocampal cultures.
(A) Cultures (8 DIV) were treated with 30 lM KA in the presence of
the indicated concentration of 5c (j). (B) Cultures (8 DIV) were
treated with the indicated concentration of KA either alone (m) or in
the presence of 1 lM (j) or 3 lM (h) 5c. Culture viability was
assessed 48 h later. Data are means SEM of three independent
experiments.
1
less oil. Rf = 0.61 (cyclohexane/diethyl ether 70:30). H
NMR (300 MHz, CDCl3): d ppm 3.17 (AB of ABX,
2H, JAB = 13.7 Hz, JAX = 8.5 Hz, JBX = 7.1 Hz,
Dm = 66.9 Hz, CH2-Ph), 3.60 (s, 3H, CH3), 3.76 (dd,
1H, J = 8.5 Hz, and J = 7.1 Hz, CH-Bn), 5.92–5.95 (m,
2H, OCH2O), 6.72 (s, 2H, Ar), 6.85 (s, 1H, Ar), 7.11
(d, 2H, J = 6.6 Hz, Ar), 7.17–7.41 (m, 3H, Ar). Anal.
Calcd for C17H16O4: C, 71.82; H, 5.67. Found: C,
71.59; H, 5.48.
4. Experimental
4.1. Chemistry
4.2.3. Methyl 2-[2-(3,5-dimethyl-4-nitrobenzoyl)-4,5-
methylenedioxyphenyl]acetate (10). 3,5-Methyl-4-nitro-
benzoic acid (331 mg, 1.69 mmol) and phosphorus pent-
oxide (2 g) were added to a stirred 1,2-dichloroethane
solution (100 mL) of 6 (253 mg, 1.3 mmol). The mixture
was further stirred at room temperature for 16 h, then
water (30 mL) was cautiously added and the mixture ex-
tracted with chloroform (2 · 30 mL). The organic layer
was separated and sequentially treated with 10% NaOH
(30 mL), brine (30 mL), and water (2 · 30 mL). The or-
ganic phase was dried (Na2SO4) and the solvent re-
moved under reduced pressure to yield crude 10 which
was purified by column chromatography using diethyl
ether/light petroleum (50:50) as eluant; pale yellow pow-
der. Mp = 127–128 ꢁC (300 mg, 62%) Rf = 0.51 (diethyl
ether/light petroleum 50:50); 1H NMR (300 MHz,
CDCl3), d ppm 2.35 (s, 6H, 2CH3), 3.64 (s, 3H,
COOCH3), 3.83 (s, 2H, CH2), 6.06 (s, 2H, OCH2O),
6.83 and 6.84 (2s, 2H, H-6 and H-3), 7.51–7.52 (m,
2H, H-20,60). Anal. Calcd for C19H17NO7: C, 61.45; H,
4.61; N, 3.77. Found: C, 61.73; H, 4.72; N, 3.52.
Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. Elemental analyses were
carried out on a C. Erba Model 1106 (elemental ana-
lyzer for C, H, and N) and the results are within
0.4% of the theoretical values. Merck silica gel 60
F254 plates were used as analytical TLC; column chro-
matography was performed on Merck silica gel 60
(70–230 mesh). 1H NMR spectra were recorded in
CDCl3 or DMSO-d6 by means of a Varian Gemini 300
spectrometer. Chemical shifts are expressed in d (ppm)
relative to TMS as internal standard and coupling con-
stants (J) in Hz. Compounds 1a–1c, 2a, 5a–5c, and 5f
were synthesized as reported in the literature.12,13,20,21
4.2. Synthesis
4.2.1. Methyl 2-(3,4-methylenedioxyphenyl)butyrate (8).
To a stirred solution of methyl 3,4-methylenedioxyphe-
nylacetate 6 (3 g, 15.45 mmol) in anhydrous THF
(50 mL) at ꢀ70 ꢁC was added potassium hydride
(682 mg, 17 mmol). After 30 min was added ethyl iodide
(1.36 mL, 2.65 g, 17 mmol), keeping the temperature at
ꢀ70 ꢁC for many hours. The cooling bath was removed
and the reaction mixture was stirred for further 16 h,
then diluted with 50% acetic acid, poured into water,
4.2.4. Methyl 2-[4,5-methylenedioxy-2-(3-methoxy-4-
nitrobenzoyl)-phenyl]acetate (11). With a similar proce-
dure, 11 was prepared from 6 (693 mg, 3.57 mmol), 3-
methoxy-4-nitrobenzoic acid (914 mg, 4.64 mmol), and

2206
N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
phosphorus pentoxide (2 g); yellow soft powder.
Mp = 152–153 ꢁC (400 mg, 30%) Rf = 0.33 (diethyl
ether/light petroleum 50:50); 1H NMR (300 MHz,
CDCl3), d ppm 3.63 (s, 3H, COOCH3), 3.84 (s, 2H,
CH2), 4.00 (s, 3H, OCH3), 6.06 (s, 2H, OCH2O), 6.84
(s, 2H, H-6 and H-3), 7.32 (dd, 1H, Jo = 8.2 Hz,
Jm = 1.4 Hz, H-60), 7.54 (d, 1 H, Jm = 1.4 Hz, H-20),
7.83 (d, 1 H, Jo = 8.2 Hz, H-50). Anal. Calcd for
C18H15NO8: C, 57.91; H, 4.05; N, 3.75. Found: C,
58.12; H, 4.21; N, 3.64.
(670 mg, 60%) Rf = 0.50 (diethyl ether/light petroleum
40:60); H NMR (300 MHz, CDCl3): d ppm 3.19 (AB
1
of
ABX,
2H,
JAB = 13.5 Hz,
JAX = 7.1 Hz,
JBX = 8.2 Hz, Dm = 58.9 Hz, CH2-Ph), 3.63 (s, 3H,
CH3), 4.48 (dd, 1H, J = 7.1;Hz and 8.2 Hz, CH-Bn),
6.03–6.09 (m, 2H, OCH2O), 6.60 (s, 1H, H-6), 7.18 (s,
1H, H-3), 7.30–7.00 (m, 5H, Ar), 7.59 (d, 2H, J = 8.8
Hz, H-20,60), 8.21 (d, 2H, J = 8.8 Hz, H-30,50). Anal.
Calcd for C24H19NO7: C, 66.51; H, 4.42; N, 3.23.
Found: C, 66.82; H, 4.38; N, 3.14.
4.2.5. Methyl 2-[4,5-methylenedioxy-2-(3-methyl-4-nitro-
benzoyl)-phenyl]propionate (12). With a similar proce-
dure, 12 was prepared from 712 (826 mg, 3.97 mmol),
3-methyl-4-nitrobenzoic acid (934 mg, 5.16 mmol), and
4.2.9. 1-(4-Amino-3,5-dimethylphenyl)-3,5-dihydro-7,8-
methylenedioxy-4H-2,3-benzodiazepin-4-one (1d). Hydra-
zine hydrate (0.13 mL, 2.59 mmol) and HCl 6N
(0.4 mL) were added to a solution of compound 10
(300 mg, 0.81 mmol) in n-butanol (40 mL), then the mix-
ture was heated at reflux and stirred for 20 h. After cool-
ing at room temperature, the formed precipitate was
filtered off, washed with MeOH, dried, and recrystallized
from MeOH to give pure nitroderivative. Data of nitro-
derivative: mp > 300 ꢁC (140 mg, 49%) Rf = 0.38
(EtOAc/cyclohexane 50:50); 1H NMR (300 MHz,
CDCl3), d ppm 2.34 (s, 6H, 2CH3), 3.46 (s, 2H, CH2),
6.05 (s, 2H, OCH2O), 6.58 (s, 1H, H-6), 6.84 (s, 1H,
H-9), 7.36 (s, 2H, H-20,60), 8.70 (br s, 1H, NH). A sus-
pension of nitroderivative (140 mg, 0.4 mmol), Raney-
Ni, and ammonium formate (100 mg, 1.58 mmol) in
EtOH (40 mL) was stirred under reflux for 2 h and then
filtered through Celite. 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, dried over
Na2SO4, was evaporated under reduced pressure and
the residue was purified by silica gel column chromatog-
raphy eluting with EtOAc/MeOH 99:1 to afford 1d as
yellow soft powder. Data of 1d: mp = 297–300 ꢁC (dec)
phosphorus
(387 mg, 26%) Rf = 0.40 (diethyl ether/light petroleum
pentoxide
(2 g).
Mp = 102–104 ꢁC
1
40:60); H NMR (300 MHz, CDCl3), d ppm 1.50 (d,
3H, J = 7.1 Hz, CH3-a), 2.63 (s, 3H, Ar-CH3), 3.60 (s,
3H, OCH3), 4.11 (q, 1H, J = 7.1 Hz, CH), 6.06 (s, 2H,
OCH2O), 6.75 (s, 1H, H-6), 7.00 (s, 1H, H-3), 7.71 (d,
1H, J = 8.2 Hz, H-60), 7.77 (s, 1H, H-20), 7.99 (d, 1H,
J = 8.2 Hz, H-50). Anal. Calcd for C19H17NO7: C,
61.45; H, 4.61; N, 3.77. Found: C, 61.26; H, 4.34; N,
3.95.
4.2.6. Methyl 2-[4,5-methylenedioxy-2-(4-nitrobenzoyl)-
phenyl]butyrate (13). With a similar procedure, 13 was
prepared from 8 (1.5 g, 6.75 mmol), 4-nitrobenzoic acid
(1.47 g, 87.7 mmol), and phosphorus pentoxide (4 g).
Mp = 179–181 ꢁC (1.2 g, 48%) Rf = 0.51 (diethyl ether/
1
light petroleum 40:60); H NMR (300 MHz, CDCl3), d
ppm 0.86 (t, 3H, J = 7.4, CH2CH3) 1.70–1.86 and
2.01–2.10 (2m, 2H, CH2CH3), 3.60 (s, 3H, OCH3),
3.90 (t, 1H, J = 7.4 Hz, CH), 6.05 (s, 2H, OCH2O),
6.72 (s, 1H, H-6), 7.06 (s, 1H, H-3), 7.94 (d, 2H,
J = 8.8 Hz, H-20,60), 8.31 (d, 2H, J = 8.8 Hz, H-30,50).
Anal. Calcd for C19H17NO7: C, 61.45; H, 4.61; N,
3.77. Found: C, 61.62; H, 4.50; N, 3.64.
1
(55 mg, 43%) Rf = 0.73 (EtOAc/MeOH 99:1). H NMR
(300 MHz, CDCl3), d ppm 2.19 (s, 6H, 2CH3), 3.42 (s,
2H, CH2), 3.86 (br s, 2H, NH2), 6.02 (s, 2H, OCH2O),
6.71 (s, 1H, H-6), 6.82 (s, 1H, H-9), 7.19 (s, 2H, H-
20,60), 8.52 (br s, 1H, NH). Anal. Calcd for
C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C,
66.67; H, 5.42; N, 13.21.
4.2.7. Methyl 2-[4,5-methylenedioxy-2-(3-methyl-4-nitro-
benzoyl)-phenyl]butyrate (14). With a similar procedure,
14 was prepared from 8 (2.8 g, 11.8 mmol), 3-methyl-4-
nitrobenzoic acid (2.8 g, 15.3 mmol), and phosphorus
pentoxide (6 g). The crude was purified by column chro-
matography using diethyl ether/light petroleum (60:40)
as eluant. Mp = 110–112 ꢁC (2.4 g, 56%) Rf = 0.6
(diethyl ether/light petroleum 60:40); 1H NMR
(300 MHz, CDCl3), d ppm 0.86 (t, 3H, J = 7.1 Hz,
CH2CH3), 1.65–2.18 (2m, 2H, CH2CH3), 2.63 (s, 3H,
Ar-CH3), 3.61 (s, 3H, OCH3), 3.86 (t, 1H, J = 6.9 Hz,
CH), 6.05 (s, 2H, OCH2O), 6.74 (s, 1H, H-6), 7.06 (s,
1H, H-3), 7.65 (dd, 1H, Jo = 8.2 Hz and Jm = 1.8 Hz,
H-60), 7.76 (d, collapsed, 1H, H-20), 7.99 (d, 1H,
J = 8.2 Hz, H-50). Anal. Calcd for C20H19NO7: C,
62.33; H, 4.97; N, 3.64. Found: C, 62.21; H, 5.17; N,
3.75.
4.2.10. 1-(4-Amino-3-methoxyphenyl)-3,5-dihydro-7,8-
methylenedioxy-4H-2,3-benzodiazepin-4-one (1e). With
a similar procedure, 1e was prepared from 11 (400 mg,
1.07 mmol), hydrazine hydrate (0.17 mL, 3.43 mmol),
and HCl 6N (0.52 mL) in n-butanol (40 mL). Data of
nitroderivative:
mp = 267–271 ꢁC
(80 mg,
21%)
Rf = 0.22 (EtOAc/cyclohexane 50:50); 1H NMR
(300 MHz, CDCl3), d ppm 3.48 (s, 2H, CH2), 4.01 (s,
3H, OCH3), 6.06 (s, 2H, OCH2O), 6.59 (s, 1H, H-6),
6.85 (s, 1H, H-9), 7.13 (d, 1H, Jo = 8.5 Hz, H-60), 7.49
(s, 1H, H-50), 7.86 (d, 1H, Jo = 8.5 Hz, H-20), 8.50 (br
s, 1H, NH). Successive reduction was performed starting
from nitro derivative (80 mg, 0.23 mmol), Raney-Ni as
catalyst, and an excess of ammonium formate. Data of
1e: mp = 247–251 ꢁC (47 mg, 64%) Rf = 0.73 (EtOAc/
MeOH 99:1); 1H NMR (300 MHz, CDCl3), d ppm:
3.43 (s, 2H, CH2), 3.90 (s, 3H, OCH3), 4.08 (br s, 2H,
NH2), 6.02 (s, 2H, OCH2O), 6.66 (d, 1H, Jo = 8.0 Hz,
H-50), 6.73 (s, 1H, H-6), 6.81 (s, 1H, H-9), 6.87 (dd,
4.2.8. Methyl 2-[4,5-methylenedioxy-2-(4-nitrobenzoyl)-
phenyl]-3-phenylpropionate (15). With a similar proce-
dure, 15 was prepared from 9 (726 mg, 3.97 mmol), 4-
nitrobenzoic acid (556 mg, 3.32 mmol), and phosphorus
pentoxide (2 g); yellow crystals. Mp = 141–143 ꢁC

N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
2207
1H, 7.13, Jo = 8.0 Hz, Jm = 1.6 Hz, H-60), 7.25 (d, 1H,
Jm = 1.9 Hz, H-20), 8.34 (br s, 1H, NH). Anal. Calcd
for C17H15N3O4: C, 62.76; H, 4.65; N, 12.92. Found:
C, 66.58; H, 4.56; N, 12.73.
CH2CH3), 2.36–2.42 (m, 1H, CH), 2.64 (s, 3H, Ar-
CH3), 6.02–6.07 (m, 2H, OCH2O), 6.56 (s, 1H, H-9),
6.90 (s, 1H, H-6), 7.85 (d, 1H, J = 8.5 Hz, H-60), 7.64
(s, 1H, H-20), 8.02 (d, 1H, J = 8.5 Hz, H-50), 8.68 (br s,
1H, NH). Successive reduction was performed starting
from nitroderivative (400 mg, 1.09 mmol), Raney-Ni as
catalyst, and an excess of ammonium formate. Data of
3c: mp = 240–242 ꢁC (360 mg, 92%) Rf = 0.3 (EtOAc/
cyclohexane 50:50). 1H NMR (300 MHz, CDCl3), d
ppm 1.08 (t, 3H, J = 7.1 Hz, CH2CH3), 1.92–2.37 (2m,
2H, CH2CH3), 2.18 (s, 3H, Ar-CH3), 2.98 (m, 1H,
CH), 3.87 (br s, 2H, NH2), 5.98–6.05 (m, 2H, OCH2O),
6.68 (d, 1H, J = 8.5 Hz, H-50), 6.71 (s, 1H, H-9), 6.82 (s,
1H, H-6), 7.28 (d, J = 8.5 Hz 1H, H-60), 7.39 (s, 1H, H-
20), 8.31 (br s, 1H, NH). Anal. Calcd for C19H19N3O3:
C, 67.64; H, 5.68; N, 12.46. Found: C, 67.49; H, 5.82;
N, 12.36.
4.2.11. 1-(4-Amino-3-methylphenyl)-3,5-dihydro-5-methyl-
7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (2c). With
a similar procedure, 2c was prepared from 12 (387 mg,
1.04 mmol), hydrazine hydrate (0.06 mL, 1.15 mmol) in
n-butanol (40 mL); yellow powder. Data of nitroderiva-
tive: mp = 273–275 ꢁC (99 mg, 27%) Rf = 0.54 (EtOAc/
cyclohexane 50:50); 1H NMR (300 MHz, CDCl3), d
ppm: 1.63 (d, 3H, J = 6.9 Hz, CH3-a), 2.64 (s, 3H, Ar-
CH3), 3.25 (q, 1H, J = 6.9 Hz, CH), 6.06 (s, 2H, OCH2O),
6.56 (s, 1H, H-9), 6.90 (s, 1H, H-6), 7.58 (d, 1H,
J = 8.5 Hz, H-60), 7.64 (s, 1H, H-20), 8.02 (d, 1H,
J = 8.5 Hz, H-50), 8.60 (br s, 1H, NH). Successive reduc-
tion was performed starting from nitroderivative (100 mg,
0.28 mmol), Raney-Ni as catalyst, and an excess of
ammonium formate. Data of 2c: mp = 239–241 ꢁC
4.2.14. 1-(4-Aminophenyl)-5-benzyl-3,5-dihydro-7,8-meth-
ylenedioxy-4H-2,3-benzodiazepin-4-one (4a). With a sim-
ilar procedure, 4a was prepared from 15 (670 mg,
1.55 mmol), hydrazine hydrate (0.24 mL, 4.95 mmol)
in n-butanol (40 mL); yellow powder. Data of nitrode-
rivative: mp = 212–216 ꢁC (72 mg, 11%) Rf = 0.54
1
(74 mg, 82%) Rf = 0.55 (EtOAc/cyclohexane 80:20); H
NMR (300 MHz, CDCl3),
d ppm: 1.59 (d, 3H,
J = 6.6 Hz, CH3-a), 2.19 (s, 3H, Ar-CH3), 3.27 (q, 1H,
J = 6.6 Hz, CH), 3.87 (br s, 2H, NH2), 6.02 (s, 2H,
OCH2O), 6.67 (d, 1H, J = 8.2 Hz, H-50), 6.70 (s, 1H, H-
9), 6.86 (s, 1H, H-6), 7.26 (d collapsed, 1H, H-60), 7.37
(s, 1H, H-20), 8.29 (br s, 1H, NH). Anal. Calcd for
C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C,
67.07; H, 5.37; N, 12.87.
1
(CH2Cl2/EtOAc 90:10); H NMR (300 MHz, CDCl3):
d ppm 3.44 (dd, 1H, J = 5.8 and 8.0 Hz, CH-Bn), 3.52
(AB of ABX, 2H, JAB = 13.7 Hz, JAX = 8.0 Hz,
JBX = 5.8 Hz, Dm = 75.8 Hz, CH2-Ph), 6.02–6.08 (m,
2H, OCH2O), 6.57 (s, 1H, H-6), 6.95 (s, 1H, H-9),
7.36–7.00 (m, 5H, Ar), 7.85 (d, 2H, J = 8.8 Hz, H-
20,60), 8.22 (d, 2H, J = 8.8 Hz, H-30,50), 8.59 (br s, 1H,
NH). Successive reduction was performed starting from
nitroderivative (72 mg, 0.17 mmol), Raney-Ni as cata-
lyst, and an excess of ammonium formate. Data of 4a:
mp = 149–153 ꢁC (30 mg, 46%) Rf = 0.56 (EtOAc/
diethyl ether/petroleum ether 60:20:20); 1H NMR
(300 MHz, CDCl3): d ppm 3.49 (dd, 1H, J = 6.0 and
8.5 Hz, CH-Bn), 3.50 (AB of ABX, 2H, JAB = 14.3 Hz,
JAX = 8.5 Hz, JBX = 6.0 Hz, Dm = 74.7 Hz, CH2-Ph),
5.95–6.04 (m, 2H, OCH2O), 6.68 (d, 2H, J = 8.5 Hz,
H-30,50), 6.71 (s, 1H, H-9), 6.90 (s, 1H, H-6), 7.36–7.00
(m, 5H, Ar), 7.45 (d, 2H, J = 8.5 Hz, H-20,60), 8.54 (br
s, 1H, NH). Anal. Calcd for C23H19N3O3: C, 71.67; H,
4.97; N, 10.90. Found: C, 71.84; H, 4.86; N, 11.07.
4.2.12. 1-(4-Aminophenyl)-3,5-dihydro-5-ethyl-7,8-meth-
ylenedioxy-4H-2,3-benzodiazepin-4-one (3a). With a sim-
ilar procedure, 3a was prepared from 13 (700 mg,
1.89 mmol), hydrazine hydrate (0.12 mL, 2.45 mmol),
and n-butanol (50 mL). Data of nitroderivative:
mp = 247–250 ꢁC (350 mg, 52%) Rf = 0.75 (EtOAc/
cyclohexane 60:40). 1H NMR (300 MHz, CDCl3), d
ppm: 1.10 (t, 3H, CH2CH3), 1.96–2.05 and 2.36–2.43
(2m, 2H, CH2CH3), 2.89–2.93 (m, 1H, CH), 6.05 (d,
2H, OCH2O), 6.55 (s, 1H, H-9), 6.85 (s, 1H, H-6),
7.85 (d, 2H, J = 8.8 Hz, H-30,50), 8.28 (d, 2H,
J = 8.8 Hz, H-20,60), 8.68 (br s, 1H, NH). Successive
reduction was performed starting from nitroderivative
(350 mg, 0.99 mmol), Raney-Ni as catalyst, and an ex-
cess of ammonium formate. Data of 3a: mp = 222–
225 ꢁC (101 mg, 31%) Rf = 0.41 (EtOAc/cyclohexane
60:40). 1H NMR (300 MHz, CDCl3), d ppm 1.07 (t,
3H, CH2CH3), 1.89–1.98 and 2.29–2.37 (2m, 2H,
CH2CH3), 2.83–2.98 (m, 1H, CH), 3.93 (br s, 2H,
NH2), 5.98–6.02 (m, 2H, OCH2O), 6.69 (d, 2H,
J = 8.5 Hz, H-30,50), 6.70 (s, 1H, H-9) 6.81 (s, 1H, H-
6), 7.46 (d, 2H, J = 8.5 Hz, H-20,60), 8.30 (br s, 1H,
NH). Anal. Calcd for C18H17N3O3: C, 66.86; H, 5.30;
N, 13.00. Found: C, 66.62; H, 5.48; N, 13.25.
4.2.15. 1-(4-Amino-3-chlorophenyl)-3,5-dihydro-5-methyl-
7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (2f). A
suspension of 16 (75 mg, 0.22 mmol) and an excess of
Raney-Ni in EtOH (30 mL) was stirred with ammonium
formate (250 mg, 4.1 mmol) to afford compound 2a as
previously described.12 NCS (28 mg, 0.21 mmol) was
added to a solution of 2a (65 mg, 0.21 mmol) in DMF
(2.5 mL) and the reaction mixture was stirred at room
temperature for 20 h. The mixture was diluted with
EtOAc (40 mL), washed with water and brine, dried
over Na2SO4, and evaporated in vacuo. The crude was
purified by a silica gel column chromatography using
EtOAc/cyclohexane 70:30 as eluant. Mp = 154–156 ꢁC
4.2.13. 1-(4-Amino-3-methylphenyl)-3,5-dihydro-5-ethyl-
7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one
(3c).
With a similar procedure, 3c was prepared from 14
(2.4 g, 6.2 mmol), hydrazine hydrate (1 mL, 20 mmol)
in EtOH (70 mL). Data of nitroderivative: mp = 280–
282 ꢁC (800 mg, 36%) Rf = 0.65 (EtOAc/cyclohexane
50:50). 1H NMR (300 MHz, CDCl3), d ppm: 1.10
(t, 3H, J = 7.1 Hz, CH2CH3), 1.94–2.35 (2m, 2H,
1
(58 mg, 80%) Rf = 0.65 (EtOAc/cyclohexane 70:30); H
NMR (300 MHz, CDCl3),
d ppm 1.59 (d, 3H,
J = 6.9 Hz, CH3-a), 3.25 (q, 1H, J = 6.9 Hz, CH), 4.33
(br s, 2H, NH2), 6.03 (m, 2H, OCH2O), 6.68 (s, 1H,
H-9), 6.77 (d, 1H, Jo = 8.2 Hz, H-50), 6.87 (s, 1H, H-

2208
N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
6), 7.35 (dd, 1H, Jo = 8.2 Hz, Jm = 1.6 Hz, H-60), 7.55
(d, 1H, Jm = 1.6 Hz, H-20), 8.50 (br s, 1H, NH). Anal.
Calcd for C17H14ClN3O3: C, 59.39; H, 4.11; N, 12.23.
Found: C, 59.57; H, 3.97; N, 12.42.
7.53 (m, 2H, H-20,60). Anal. Calcd for C20H19NO7: C,
62.33; H, 4.97; N, 3.64. Found: C, 62.27; H, 4.89; N,
3.71.
4.2.19. Methyl 2-[4,5-ethylenedioxy-2-(3-methoxy-4-
nitrobenzoyl)-phenyl]propionate (20). With a similar pro-
cedure, 20 was prepared from 1713 (345 mg, 1.67 mmol),
3-methoxy-4-nitrobenzoic acid (427 mg, 2.17 mmol),
and phosphorus pentoxide (2 g); light yellow crystals.
Mp = 156–158 ꢁC (400 mg, 62%) Rf = 0.37 (diethyl
ether/light petroleum 50:50); 1H NMR (300 MHz,
CDCl3), d ppm 3.64 (s, 3H, COOCH3), 3.86 (s, 2H,
CH2), 4.00 (s, 3H, OCH3), 4.24–4.37 (m, 4H, OCH2-
CH2O), 6.86 (s, 1H, H-60), 6.94 (s, 1H, H-30), 7.33 (dd,
1H, Jo = 8.2 Hz, Jm = 1.6 Hz, H-60), 7.54 (d, 1H,
Jm = 1.6 Hz, H-20), 7.84 (d, 1H, Jo = 8.2 Hz, H-50).
Anal. Calcd for C19H17NO8: C, 58.91; H, 4.42; N,
3.62. Found: C, 58.78; H, 4.57; N, 3.49.
4.2.16. 1-(4-Aminophenyl)-3,5-dihydro-5-methyl-3-N-meth-
ylcarbamoyl-7,8-methylendioxy-4H-2,3-benzodiazepin-4-
one (2b). To a solution of 1612 (300 mg, 0.88 mmol) in
CH2Cl2 (50 mL) were added triethylamine (1.15 mL,
8.23 mmol) and methyl isocyanate (253 mg, 4.42 mmol).
The reaction mixture was stirred at room temperature
for 24 h and then washed with slightly acid solution
and brine, and extracted with chloroform. The organic
phase was dried over Na2SO4 and the solvent removed
under reduced pressure. The resulting residue was puri-
fied by treatment with diethyl ether and used at the next
step without further purification. The subsequent hydro-
genation was carried out at atmospheric pressure by
adding 5% Pd/C (50 mg) to a chloroformic solution
(50 mL) of the nitro derivate. The mixture was shaken
under hydrogen for 3 h and the Pd/C was filtered out
through a Celite pad. The solution was washed with
water and brine, and extracted with CHCl3. The com-
bined organic layers were dried over Na2SO4, evapo-
rated under reduced pressure, and the residue was
purified by silica gel column chromatography (CHCl3/
EtOAc 50:50) to afford 2b as a white solid. Further puri-
fication was obtained by trituration of the powder with
ethyl ether. Mp = 186–188 ꢁC (109 mg, 34%) Rf = 0.35
4.2.20. Methyl 2-[4,5-ethylenedioxy-2-(4-nitrobenzoyl)-
phenyl]propionate (21). With a similar procedure, 21
was prepared from 18 (1.07 g, 4.8 mmol), 4-nitrobenzoic
acid (1.02 g, 6.3 mmol), and phosphorus pentoxide (2 g).
The product was purified by silica gel column chroma-
tography (cyclohexane/EtOAc 70:30) Mp = 113–115 ꢁC
(841 mg, 48%) Rf = 0.38 (cyclohexane/EtOAc 70:30);
1H NMR (300 MHz, CDCl3), d ppm 1.52 (d, 3H,
J = 7.2 Hz, CH3-a), 3.59 (s, 3H, OCH3), 4.22–4.34 (m,
5H, CH and OCH2CH2O) 6.84 (s, 1H, H-6), 7.01 (s,
1H, H-3), 7.94 (d, 2H, J = 8.8 Hz, H-20,60), 8.30 (d,
2H, J = 8.8 Hz, H-30,50). Anal. Calcd for C19H17NO7:
C, 61.45; H, 4.61; N, 3.77. Found: C, 61.28; H, 4.57;
N, 3.91.
1
(CHCl3/EtOAc 50:50); H NMR (300 MHz, CDCl3), d
ppm 1.56 (d, 3H, J = 6.6 Hz, CH3-a), 2.89 (d, 3H,
J = 4.7 Hz, NHCH3), 3.49 (q, 1H, J= 6.6 Hz, CH),
3.97 (br s, 2H, NH2), 6.00–6.07 (m, 2H, OCH2O), 6.68
(d, 2H, J = 8.8 Hz, H-30,50), 6.72 (s, 1H, H-9), 6.86 (s,
1H, H-6), 7.58 (d, 2H, J = 8.8 Hz, H-20,60), 8.66 (q col-
lapsed, 1H, NHCH3). Anal. Calcd for C19H19N4O4: C,
62.28; H, 4.95; N, 15.29. Found: C, 62.41; H, 4.78; N,
15.18.
4.2.21. Methyl 2-[4,5-ethylenedioxy-2-(3-methyl-4-nitro-
benzoyl)phenyl]propionate (22). With a similar proce-
dure, 22 was prepared from 18 (1.23 g, 5.6 mmol), 3-
methyl-4-nitrobenzoic acid (1.31 g, 7.2 mmol), and
phosphorus pentoxide (2 g). The product was purified
by silica gel column chromatography (diethyl ether/light
petroleum 60:40). Mp = 117–119 ꢁC (858 mg, 49%)
Rf = 0.53 (diethyl ether/light petroleum 60:40); 1H
4.2.17. Methyl 2-(3,4-ethylenedioxyphenyl)propionate
(18). With a procedure similar to that reported for 7,12
18 was prepared from methyl 3,4-ethylenedioxypheny-
lacetate 1713 (1.85 g, 9 mmol), potassium hydride
(400 mg, 10 mmol), and methyl iodide (1.4 g, 10 mmol)
in anhydrous THF (40 mL). Compound 18 was isolated
as colorless oil (1.07 g, 54%) by silica gel column chro-
matography (cyclohexane/EtOAc 70:30). Rf = 0.54
(cyclohexane/EtOAc 70:30). 1H NMR (300 MHz,
CDCl3), d ppm 1.47 (d, 3H, J = 6.9 Hz, CH3-a), 3.66
(m, 4H, CH and OCH3), 4.22 (s, 4H, OCH2CH2O),
6.79–4–6.84 (m, 3H, Ar). Anal. Calcd for C12H14O4:
C, 64.85; H, 6.35. Found: C, 64.65; H, 6.52.
NMR (300 MHz, CDCl3),
d ppm 1.51 (d, 3H,
J = 7.1 Hz, CH3-a), 2.63 (s, 3H, Ar-CH3), 3.68 (s, 3H,
OCH3), 4.15–4.35 (m, 5H, CH and OCH2CH2O) 6.85
(s, 1H, H-6), 7.01 (s, 1H, H-3), 7.70 (dd, Jo = 8.2 Hz
and Jm = 1.6 Hz, H-60), 7.76 (d collapsed, 1H, H-20),
7.99 (d, 1H, Jo = 8.5 Hz, H-50). Anal. Calcd for
C20H19NO7: C, 62.33; H, 4.97; N, 3.64. Found: C,
62.52; H, 4.81; N, 3.77.
4.2.22. 1-(4-Amino-3,5-dimethylphenyl)-3,5-dihydro-7,8-
ethylenedioxy-4H-2,3-benzodiazepin-4-one (5d). With a
procedure similar to that reported for 1d, 5d was pre-
pared from 19 (300 mg, 0.78 mmol), hydrazine hydrate
(0.12 mL, 2.49 mmol), and HCl 6 N (0.38 mL). Data
of nitroderivative: mp > 300 ꢁC (200 mg, 70%)
Rf = 0.34 (EtOAc/cyclohexane 50:50); 1H NMR
(300 MHz, CDCl3), d ppm 2.34 (s, 6H, 2CH3), 3.47 (s,
2H, CH2), 4.24–4.34 (m, 4H, OCH2CH2O), 6.67 (s,
1H, H-5), 6.89 (s, 1H, H-9), 7.37 (s, 2H, H-20 and H-
60), 8.56 (br s, 1H, NH). Successive reduction was per-
formed starting from nitroderivative (200 mg,
4.2.18. Methyl 2-[2-(3,5-dimethyl-4-nitrobenzoyl)-4,5-eth-
ylenedioxy-phenyl]propionate (19). With a procedure
similar to that reported for 10, 19 was prepared from
1713 (201 mg, 0.97 mmol), 3,5-methyl-4-nitrobenzoic
acid (247 mg, 1.26 mmol), and phosphorus pentoxide
(2 g). White crystals. Mp = 143–144 ꢁC (300 mg, 80%)
Rf = 0.38 (diethyl ether/light petroleum 50:50); 1H
NMR (300 MHz, CDCl3), d ppm 2.35 (s, 6H, 2ArCH3)
3.62 (s, 3H, COOCH3), 3.84 (s, 2H, CH2), 4.27–4.32 (m,
4H, OCH2CH2O), 6.85 (s, 1H, H-6), 6.94 (s, 1H, H-3),

N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
2209
0.55 mmol), Raney-Ni as catalyst, and an excess of
ammonium formate. Data of 5d: mp = 239–242 ꢁC
(dec) (50 mg, 27%) Rf = 0.69 (EtOAc/MeOH 99:1); H
NMR (300 MHz, CDCl3), d ppm 2.18 (s, 6H, 2CH3),
3.42 (s, 2H, CH2), 3.85 (br s, 2H, NH2), 4.22–4.34 (m,
4H, OCH2CH2O), 6.80 (s, 1H, H-5), 6.85 (s, 1H, H-8),
7.20 (s, 2H, H-20,60), 8.48 (br s, 1H, NH). Anal. Calcd
for C19H19N3O3: C, 67.64; H, 5.68; N, 12.46. Found:
C, 67.38; H, 5.74; N, 12.52.
7.1 mmol). Data of nitroderivative: mp = 259–260 ꢁC
(165 mg, 20%) Rf= 0.78 (CH2Cl2/EtOH 95:5); 1H
NMR (DMSO-d6): 1.41 (d, 3H, J = 6.3 Hz, CH3-a),
2.54 (s, 3H, Ar-CH3), 3.20–3.32 (m, 1H, CH), 4.22–
4.30 (m, 4H, OCH2CH2O), 6.59 (s, 1H, H-9), 6.84 (s,
1H, H-6), 7.56 (d, 1H, J = 8.5 Hz, H-60), 7.63 (s, 1H,
H-20), 8.06 (d, 1H, J = 8.5 Hz, H-50), 11.16 (br s, 1H,
NH). Successive reduction was performed starting from
nitro derivative (165 mg, 0.45 mmol), Raney-Ni as cata-
lyst, and an excess of ammonium formate. Data of 6c:
Mp = 260–263 ꢁC (60 mg, 36%) Rf = 0.5 (CH2Cl2/EtOH
1
4.2.23. 1-(4-Amino-3-methoxyphenyl)-3,5-dihydro-7,8-
ethylenedioxy-4H-2,3-benzodiazepin-4-one (5e). With a
procedure similar to that reported for 1e, 5e was pre-
pared from 20 (400 mg, 1.03 mmol), hydrazine hydrate
(0.16 mL, 3.30 mmol), and HCl 6 N (0.5 mL). Data of
nitroderivative: Mp = 234–236 ꢁC (156 mg, 41%)
Rf = 0.22 (EtOAc/cyclohexane 50:50); 1H NMR
(300 MHz, CDCl3), d ppm 3.49 (s, 2H, CH2), 4.01 (s,
3H, OCH3), 4.19–4.41 (m, 4H, OCH2CH2O), 6.68 (s,
1H, H-6), 6.89 (s, 1H, H-9), 7.13 (dd, 1H, Jo = 8.5,
Jm = 1.6, H-60), 7.50 (d, 1H, Jm = 1.6, H-50), 7.86 (d,
1H, Jo = 8.5, H-20), 8.62 (br s, 1H, NH). Successive
reduction was performed starting from nitro derivative
(156 mg, 0.42 mmol), Raney-Ni as catalyst, and an ex-
cess of ammonium formate. Data of 5e: mp = 250–
252 ꢁC (76 mg, 53%) Rf = 0.69 (EtOAc/MeOH 99:1);
1H NMR (300 MHz, CDCl3), d ppm 3.43 (s, 2H,
CH2), 3.90 (s, 3H, OCH3), 4.09 (br s, 2H, NH2), 4.22–
4.34 (m, 4H, OCH2CH2O), 6.66 (d, 1H, Jo = 8.0 Hz,
H-50), 6.82 (s, 1H, H-6), 6.85 (s, 1H, H-9), 6.87 (dd,
1H, Jo= 8.0 Hz, Jm = 1.6 Hz, H-60), 7.26 (d, 1H, Jm=
1.9 Hz, H-20), 8.31 (br s, 1H, NH). Anal. Calcd for
C18H17N3O4: C, 63.71; H, 5.05; N, 12.38. Found: C,
63.56; H, 5.18; N, 12.41.
1
95:5); H NMR (300 MHz, CDCl3), d ppm 1.57 (d, 3H,
J = 5.5 Hz, CH3-a), 2.18 (s, 3H, Ar-CH3), 3.24–3.37 (m,
1H, CH), 3,86 (br s, 2H, NH2), 4.24–4.32 (m, 4H,
OCH2CH2O), 6.66 (d, 1H, J = 8.2 Hz, H-50), 6.8 (s,
1H, H-9), 6.87 (s, 1H, H-6), 7.25 (d, J = 8.2 Hz 1H, H-
60), 7.39 (s, 1H, H-20), 8.23 (br s, 1H, NH). Anal. Calcd
for C19H19N3O3: C, 67.64; H, 5.68; N, 12.46. Found: C,
67.78; H, 5.81; N, 12.27.
4.2.26. 1-(4-Amino-3-chlorophenyl)-3,5-dihydro-7,8-eth-
ylenedioxy-5-methyl-4H-2,3-benzodiazepin-4-one
(6f).
With a procedure similar to that reported for 2f, 6f
was obtained starting from 6a (110 mg, 0.34 mmol)
and NCS (46 mg, 0.34 mmol). Mp = 148–151 ꢁC
(40 mg, 36%) Rf = 0.64 (diethyl ether/light petroleum
90:10) 1H NMR (300 MHz, CDCl3), d ppm 1.57 (d,
3H, J = 5.5 Hz, CH3-a), 3.22–3.31 (m, 1H, CH), 4.26–
4.32 (m, 4H, OCH2CH2O), 4.71 (br s, 2H, NH2), 7.41–
7.63 (m, 3H, Ar), 6.76 (s, 1H, H-9), 6.89 (s, 1H, H-6),
8.40 (br s, 1H, NH). Anal. Calcd for C18H16ClN3O3:
C, 60.40; H, 4.53; N, 11.74. Found: C, 60.28; H, 4.67;
N, 11.95.
4.3. Pharmacology
4.2.24. 1-(4-Aminophenyl)-3,5-dihydro-7,8-ethylenedioxy-
5-methyl-4H-2,3-benzodiazepin-4-one (6a). With a proce-
dure similar to that reported for 2c, 6a was prepared
from 21 (841 mg, 2 mmol), hydrazine hydrate
4.3.1. Animals. Procedures involving animals and their
care were conducted in conformity with the Institutional
Guidelines that comply with national (D.L. n.116, G.U.,
suppl. 40 Feb. 18, 1992) and international (EEC Council
Directive 86/609, OJL 358, 1, Dec. 12, 1987; NIH Guide
for Care and Use of Laboratory Animals, U.S. National
Research Council, 1996) laws and policies.
(0.12 mL,
2 mmol).
Data
of
nitroderivative:
mp > 280 ꢁC (150 mg, 18%) Rf = 0.51 (cyclohexane/
EtOAc 50:50); 1H NMR (DMSO-d6): 1.45 (d, 3H,
J = 6.3 Hz, CH3-a), 3.31 (q, 1H, J = 6.3 Hz, CH),
4.28–4.34 (m, 4H, OCH2CH2O), 6.63 (s, 1H, H-9),
6.89 (s, 1H, H-6), 7.83 (d, 2H, J = 8.3 Hz, H-20,60),
8.33 (d, 2H, J = 8.3 Hz, H-30,50), 11.25 (br s, 1H, NH).
Successive reduction was performed starting from nitro
derivative (150 mg, 0.42 mmol), Raney-Ni as catalyst,
and an excess of ammonium formate. Data of 6a:
mp > 280 ꢁC (31 mg, 24%) Rf = 0.48 (EtOAc/cyclohex-
4.3.2. [3H]CP-526,427 binding14. The binding of [3H]CP-
526,427 was characterized in rat forebrain membranes.
Forebrains of adult male Sprague–Dawley rats were
homogenized in 0.32 M sucrose at 4 ꢁC. The crude nu-
clear pellet was removed by centrifugation at 1000g for
10 min, and the supernatant centrifuged at 17,000g for
25 min. The resulting pellet was re-suspended in 5 mM
Tris–acetate, pH 7.4, at 4 ꢁC for 10 min to lyse cellular
particles and again centrifuged at 17,000g. The resulting
pellet was washed twice in Tris–acetate, re-suspended at
10 mg of protein/ml, and stored at ꢀ20 ꢁC until use.
Immediately before binding assays, membranes were
thawed, homogenized, and diluted to 0.5 mg of pro-
tein/mL with 50 mM TrisÆHCl, pH 7.4. For competition
assays, compounds were added at various concentra-
tions followed by 3 nM [3H]CP-526,427 (specific activ-
ity, 24.36 Ci/mmol). After incubation for 20 min at
30 ꢁC in a shaking water bath, samples were filtered
onto Whatman GFB glass fiber filters using a MB-48R
1
ane 70:30); H NMR (300 MHz, CDCl3), d ppm 1.56
(d, 3H, J = 6.9 Hz, CH3-a), 3.30 (q, 1H, J = 6.9 Hz,
CH), 3,85 (br s, 2H, NH2), 4.18–4.31 (m, 4H, OCH2-
CH2O), 6.67 (d, 2H, J = 8.8 Hz, H-30,50), 6.79 (s, 1H,
H-9), 6.86 (s, 1H, H-6), 7.43 (d, 2H, J = 8.8 Hz, H-
20,60), 8.57 (br s, 1H, NH). Anal. Calcd for
C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C,
67.08; H, 5.13; N, 13.18.
4.2.25. 1-(4-Amino-3-methylphenyl)-3,5-dihydro-7,8-eth-
ylenedioxy-5-methyl-4H-2,3-benzodiazepin-4-one (6c).
With a similar procedure, 6c was prepared from 22
(858 mg, 2.2 mmol), hydrazine hydrate (0.34 mL,

2210
N. Micale et al. / Bioorg. Med. Chem. 16 (2008) 2200–2211
Cell Harvester (Brandel Research and Development
Laboratories, Gaithersburg MD). Filters were washed
for 10 s with ice-cold TrisÆHCl buffer and the radioactiv-
ity trapped on the filter quantified by liquid scintillation
counting. Nonspecific binding for [3H]CP-526,427 was
determined in parallel incubations containing 10 lM
unlabeled CP-526,427 or CP-465,022. Specific binding
was defined as total binding minus nonspecific binding.
were plated at a density of 2 · 105 cells/well on poly-L-
lysine (Sigma, Milan, Italy) coated 12-well plates. Cul-
tures were grown at 37 ꢁC in a humidified incubator
with 5% CO2. The same medium (without glutamate)
was added on fourth and sixth day in vitro (DIV). Cul-
tures prepared by this method were enriched in hippo-
campal
neurons,
as
assessed
by
GFAP
immunocytochemistry (data not shown). At 7th DIV
drugs were dissolved in CCM without glutamate and
neurons were exposed for 48 h at 37 ꢁC in a humidified
incubator with 5% CO2. Neuronal survival was assayed
by measuring conversion of yellow water-soluble tetra-
zolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide—MTT, Sigma, Milan, Italy) to
blue water-insoluble formazan. Culture medium was re-
moved and cells were incubated for 4 h with 10% MTT
solution (5 mg/mL in NaCl) in fresh CCM at 37 ꢁC.
After incubation media were removed, cells were dis-
solved in dimethylsulfoxide (DMSO, Sigma, Milan,
Italy), and solution was transferred to a 96-well plate
to measure absorbance (570 nm). Viability is expressed
as a percentage of absorbance measured in untreated
cells.
4.3.3. Primary cultures of rat cerebellar granule neurons.
Primary cultures of rat cerebellar granule neurons were
prepared as described previously.16
4.3.4. 45Ca2+ uptake14. Neurons in poly-D-lysine-coated
96-well plates were preincubated for 30 min with differ-
ent concentrations of compounds in balanced salt solu-
tion (BSS; 115 mM NaCl, 5.4 mM KCl, 0.96 mM
NaH2PO4, 1.8 mM CaCl2, 11 mM d-glucose, and
25 mM Hepes, pH 7.3). They were then exposed at room
temperature to 100 lM kainate or NMDA in BSS con-
taining 10 lM glycine, 0.5 mM dithiothreitol, and
0.5 lCi 45Ca2+ (final specific activity, 2.78 lCi/lmol) in
a volume of 100 ll/well. After 10 min, the neurons were
then rapidly washed five times with 200 ll/well of ice-
cold BSS containing 5 mM EGTA. Neurons were then
lysed in 30 ll/well of 0.6% Triton X-100 and radioactiv-
ity in aliquots of the lysate was measured with a Top-
Count microtiter scintillation counter (Packard
Instrument Co., Downers Grove, IL).
Acknowledgments
We thank Dr. F.S. Menniti, Pfizer Global Research and
Development, Groton, Connecticut, USA, for supplying
with [3H]CP-526,427 radioligand, and Dr. M. De Paola
(IRFMN) for mouse hippocampal culture setting. This
work was financially supported by the Ministero dell’Ist-
4.3.5. Measurement of [Ca2+ 14. Neurons in 96-well,
]
i
black/clear, poly-D-lysine-coated tissue culture plates
were rinsed once with BSS and then incubated for 1 h
in BSS containing 4 lM Fluo-4/AM (Molecular Probes,
Inc., Eugene, OR). Fluo-4/AM was prepared immedi-
ately before use as a 1 mM stock solution in dimethyl-
sulfoxide with 10% (w/v) pluronic acid. Cells were then
washed three times and held in BSS at room tempera-
ture and used within 1 h. A fluorescent imaging plate
reader (FLIPR; Molecular Devices, Sunnyvale, CA)
was used for simultaneous imaging and fluid addition.
Cells were preincubated with test compounds for
approximately 6 min, then stimulated with 32 lM
AMPA. Changes in fluorescent intensity were measured
at a frequency of 1 sample/2 s after AMPA addition.
Raw data are expressed in relative fluorescent units
(RFUs) after the background fluorescence was
subtracted.
`
ruzione, dell’Universita e della Ricerca (MIUR-CO-
FIN2005)-Rome, Italy.
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