Synthesis, chiral resolution, and enantiopharmacology of a potent 2,3-benzodiazepine derivative as noncompetitive AMPA receptor antagonist

Article abstract of DOI:10.1021/jm050552y

This paper describes the synthesis of racemic 3,5-dihydro-5-methyl-7,8- methylenedioxy-4H-2,3-benzodiazepin-4-one (±)-5, attempted stereoselective synthesis of its enantiomers, chiral HPLC resolution of the racemate, and assignment of the absolute configuration. Enantiomer (5S)-(-)-5 is provided with an in vivo unticonvulsant activity 8 times higher than its enantiomer (5R)-(+)-5. This result is confirmed in the in vitro test by the ability to inhibit the kainate-induced increase of the [Ca²⁺] _i in a primary culture of rat cerebellar granule cells which express α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. Binding affinity of compound (±)-5 at the AMPA and N-methyl-D-aspartic acid (NMDA) receptors was also evaluated.

Full text of DOI:10.1021/jm050552y

J. Med. Chem. 2006, 49, 575-581
575
Synthesis, Chiral Resolution, and Enantiopharmacology of a Potent 2,3-Benzodiazepine
Derivative as Noncompetitive AMPA Receptor Antagonist
Maria Zappala`,∇ Giovanna Postorino,∇ Nicola Micale,*,∇ Salvatore Caccamese,# Nunziatina Parrinello,# Giovanni Grazioso,
Gabriella Roda, Frank S. Menniti,‡ Giovambattista De Sarro,§ and Silvana Grasso∇
Dipartimento Farmaco-Chimico, UniVersita` di Messina, Viale Annunziata, 98168 Messina, Italy, Dipartimento di Scienze Chimiche,
UniVersita` di Catania, Viale A. Doria 6, 95125 Catania, Italy, Istituto di Chimica Farmaceutica, UniVersita` di Milano, Viale Abruzzi 42,
20131 Milano, Italy, Pfizer Global Research and DeVelopment, Eastern Point Road, Groton, Connecticut 06340, Dipartimento di Medicina
Sperimentale e Clinica, UniVersita` di Catanzaro, Via T. Campanella, 88100 Catanzaro, Italy
ReceiVed June 13, 2005
This paper describes the synthesis of racemic 3,5-dihydro-5-methyl-7,8-methylenedioxy-4H-2,3-benzodi-
azepin-4-one (()-5, attempted stereoselective synthesis of its enantiomers, chiral HPLC resolution of the
racemate, and assignment of the absolute configuration. Enantiomer (5S)-(-)-5 is provided with an in vivo
anticonvulsant activity 8 times higher than its enantiomer (5R)-(+)-5. This result is confirmed in the in
vitro test by the ability to inhibit the kainate-induced increase of the [Ca2+]i in a primary culture of rat
cerebellar granule cells which express R-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)
receptors. Binding affinity of compound (()-5 at the AMPA and N-methyl-D-aspartic acid (NMDA) receptors
was also evaluated.
Chart 1
Introduction
Evidence suggests that glutamate, the major excitatory
neurotransmitter in the central nervous system, is involved in
several neurodegenerative syndromes, including brain ischemia
and epilepsy. In the past few years there has been considerable
interest in noncompetitive R-amino-3-hydroxy-5-methyl-4-isox-
azole propionic acid (AMPA) receptor antagonists since pro-
totype compounds, e.g. 1-(4-aminophenyl)-4-methyl-7,8-meth-
ylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466) (Chart 1),
have demonstrated significant anticonvulsant and neuroprotec-
tive action, thus providing the basis for extensive research in
this area.1-3
Highly active analogues of 1 have been found, e.g. 3,4-
dihydro-3-N-methylcarbamoyl [(()-2, GYKI 53655] and 3,4-
dihydro-3-N-acetyl [(()-3, GYKI 53405] derivatives (Chart 1).4
The enantioselectivity of derivatives (()-2 and (()-3 has been
evaluated, and the eutomer turned out to be the (4R)-enantio-
mer.3 Subsequently, the eutomer (4R)-(+)-3 was chosen as a
drug candidate and is now in clinical investigation as an
anticonvulsant and antiischemic (LY 300164, Talampanel).5
We have previously investigated6,7 a series of 1-aryl-3,5-
dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones, e.g.
4 (Chart 1), which have been shown to possess remarkable
anticonvulsant properties and are endowed with a potency higher
than that of 1. In this context we noticed that the substitution
of the iminohydrazone portion of the diazepine nucleus of 1 by
the iminohydrazide moiety increases the anticonvulsant activity.
Moreover, we have recently demonstrated that derivative 4
interacts with the same allosteric AMPA receptor binding site
of 1.8
to the allosteric AMPA binding site, has recently derived two
four-point pharmacophore models.9 Common features of these
models are (i) the electron donor amino group, (ii) the 1-phenyl
ring as hydrophobic moiety, and (iii) a second electron-donating
group which is the ether moiety attached to C-8. Among the
two models, the difference resides in the fourth attachment point.
Specifically, while in one model the fourth interaction point
involves the lone pair of 1 N-3 atom, in the other one the lone
pairs of the C-4 carbonyl oxygen of 4 are taken into account.
(4R)-(+)-3 can fit both models due to the rotating 3N-C bond.
The authors assume that the second pharmacophore model is
more appropriate, since a polar group at C-4, in replacement of
the alkyl group, gives a stronger and energetically more
pronounced interaction with the binding protein; as a conse-
quence, compounds which meet this requirement, e.g. (4R)-
(+)-3 and 4, possess a higher affinity.
A group of scientists from the IVAX Drug Research Institute,
using a set of 2,3-benzodiazepines provided with high affinity
* To whom correspondence should be addressed. Phone: +39 090
6766466. Fax: +39 090 355613. E-mail: nmicale@pharma.unime.it.
∇ Universita` di Messina.
# Universita` di Catania.
Universita` di Milano.
‡ Pfizer Global Research and Development.
§ Universita` di Catanzaro.
10.1021/jm050552y CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/16/2005

576 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Zappala` et al.
Scheme 1a
a Reagents and conditions:21 (a) MeOH, HClg, rt, 30 min; (b) KH, THF anhydrous, 30 min, -70 °C, then CH3I, -70 °C, rt, 16 h; (c) 4-nitrobenzoic acid,
P2O5, (CH2Cl)2, rt, 16 h; (d) NH2NH2, n-BuOH, reflux, 24 h; (e) Raney-Ni, ammonium formate, EtOH, reflux, 2 h; (f) carboxylesterase NP, phosphate, pH
7.0.
Considering that the carbonyl group at C-4, which represents
a steady point of our ongoing studies, characterizes the most
promising pharmacophore model, we planned an extension of
our investigations on the structure-activity relationships of 2,3-
benzodiazepin-4-ones by introducing a lipophilic substituent at
C-5 of compound 4. Its effect on the anticonvulsant activity as
well as on the affinity for AMPA receptors was consequently
checked. Now we report the synthesis of 3,5-dihydro-5-methyl-
7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (()-5 (Chart
1). Due to the different pharmacological profile previously
observed in the enantiopure forms of 3, we attempted the
stereoselective synthesis of enantiomers (+)-5 and (-)-5. Since
the chemical approach failed, we resorted to chiral HPLC to
isolate sizable quantities of single enantiomers of (()-5 and to
assign the absolute configuration. In this way, we were able to
characterize their in vivo and in vitro pharmacological profile
and to evaluate their enantiopharmacology.
been performed, at analytical scale, with a number of com-
mercially available enzymes, i.e. lipase A and lipase B from
Candida Antarctica, papain from Carica papaya, acylase I from
Aspergillus melleus, subtilisin Carlsberg, lipase PS from
Pseudomonas cepacia, and R-chymotrypsin. Among the tested
enzymes, none evidenced any discrimination among the enan-
tiomers of (()-9 while carboxylesterase NP gave a partial
resolution of substrate (()-8. The progress of the enzymatic
hydrolysis was monitored by HPLC using an analytical polysac-
charide-derived chiral column (Chiralcel OD-H, cellulose tris-
[3,5-dimethylphenylcarbamate]). The best results were obtained
when the biocatalyzed hydrolysis was stopped at 40% conver-
sion to yield acid (+)-12 (e.e. 83.2%) and unreacted ester (-)-8
(e.e. 57.2%) (Scheme 1). The stereochemistry of (+)-12 was
assigned as S, according to literature data.10 Although the
enantiomeric excess of R-(-)-8 and S-(+)-12 was not com-
pletely satisfactory, we decided to proceed with the subsequent
steps of the synthetic plan (Scheme 1) with the objective to
determine the absolute configuration of the enantiomers (+)-5
and (-)-5. Unfortunately the Friedel-Craft acylation and the
subsequent cyclization step provoked the complete racemization
of the substrate as evidenced by the chiral HPLC analyses (vide
infra) carried out on final compounds.
Results and Discussion
Chemistry. The synthesis of (()-5 was carried out following
the reaction sequence reported in Scheme 1. Commercially
available 3,4-methylenedioxyphenylacetic acid (6) was trans-
formed into methyl ester (7) and then converted in high yield
into methyl 3,4-methylenedioxy-R-phenylpropionate (8) by
R-alkylation conducted in THF at -70 °C in the presence of
potassium hydride and methyl iodide. Ketoester 9 was prepared
via acylation of 8 with 4-nitrobenzoic acid in the presence of
an excess of phosphorus pentoxide. The subsequent treatment
with hydrazine gave 3,5-dihydro-5-methyl-7,8-methylenedioxy-
1-(4-nitrophenyl)-4H-2,3-benzodiazepin-4-one (10) in low yield.
In this reaction, 2-amino-4-methyl-6,7-methylenedioxy-1-(4-
nitrophenyl)-2H-isoquinolin-3-one (11) has also been isolated
as byproduct. Reduction of the nitro group of 10, carried out
with Raney-Ni/ammonium formate, afforded derivative (()-5.
Physical and spectral data (1H and 13C NMR) of the synthesized
compounds are in agreement with the proposed structures.
Therefore, to get the pure enantiomers of (()-5 we carried
out a preparative HPLC analysis using a polysaccharide-derived
chiral stationary phase (Chiralpak AS-H, amylose tris[(S)-R-
methylbenzylcarbamate]). The resolution of (()-5 was optimized
by varying the amount of the alcohol (the modifier). As shown
in Table 1, the separation factor R is slightly affected by the
polarity of the mobile phase and the resolution factor Rs
remained high in all experiments. Parts a and b of Figure 1
show the HPLC chromatograms of experiments 1 and 5,
respectively. On the basis of the chromatographic results, the
preparative isolation of the enantiomers of compound (()-5 was
carried out using a 1:1 mixture of n-hexane/ethanol as the mobile
phase (experiment 5, Table 1). These experimental conditions
ensured, in fact, reasonable elution times and a still good Rs
factor. The analytical control performed on the first collected
fraction gave an enantiomeric excess of 100% whereas the e.e.
To accomplish the synthesis of enantiomers (+)-5 and (-)-
5, we planned the enantioselective enzyme-catalyzed hydrolysis
of esters (()-8 and ketoester (()-9. Several experiments have

Synthesis of a 2,3-Benzodiazepine DeriVatiVe
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 577
Table 1. Separation in Chiral HPLC of Compound (()-5 Using
Chiralpak AS-H Column
b
c
exp.
A (%)a
k1
k2
Rd
Rse
1
2
3
4
5f
20
25
35
40
50f
12.7
8.3
4.4
3.3
2.5
19.3
12.7
6.8
5.1
3.9
1.51
1.53
1.53
1.52
1.55
4.2
4.0
3.0
3.2
2.8
a Percentage of ethanol in n-hexane. b Capacity factor of the first
enantiomer. c Capacity factor of the second enantiomer. d Separation factor.
e Resolution factor. f Experimental conditions used for semipreparative
isolation.
Figure 3. Three-dimensional plots of the global energy minimum
conformations of compound (5R)-5 as optimized at the DFT/B3LYP/
6-31(d) level and recalculated in ethanol using the PCM model. (A)
Conformer (5R)-5A; the torsion angle optimized by PES is highlighted.
(B) Conformer (5R)-5B.
Figure 1. (a) Chromatogram referred to in conditions of experiment
1. (b) chromatogram referred to in conditions of experiment 5, used
for semipreparative isolation.
The [R]D theoretical prediction of the conformational minimized
stereoisomer (5R)-5 was carried out by Gaussian0313 software
by means of TDDFT/B3LYP/6-31G(d) as basis set.14 The
outcome of this research reveals that the 5-methyl- 2,3-
benzodiazepine ring exists, at room temperature, in two stable
conformations (Figure 3) depending on the equatorial or axial
orientation of its methyl group. Moreover, rotation of the
4-aminophenyl moiety linked at position 1 of the 2,3-benzodi-
azepine ring introduces a further conformational freedom that
had to be optimized in order to obtain the most energetically
favored conformations. Considering this torsion angle, a 360°
potential energy scan (PES) was carried out (Figure 4) on both
the ring conformers; the spatial arrangements corresponding to
the lowest potential energy were re-optimized by a conjugate
gradient method at DFT/b3lyp/ 6-31G(d) level. Only two spatial
arrangements, one for each ring conformer, were found to hold
the lowest energy potential both in a vacuum and at room
temperature. Additionally, to reproduce the experimental condi-
tions used in the [R]D estimation, the population analysis was
completed considering the PCM15 ethanol solvent model (Table
2). The calculated [R]D values of the two different spatial
arrangements of (5R)-5 were weight averaged to give an [R]D
) +655.6, a value in reasonable agreement with the experi-
mental result of +447.0. As a consequence, the R configuration
has to be assigned to enantiomer (+)-5, which is the HPLC
first-eluted isomer.
Figure 2. CD spectra of the enantiomers (+)-5 and (-)-5, in ethanol
at 22 °C.
of the second fraction amounted to 97.7%. Specific rotation of
the first-eluted enantiomer was [R]22D ) +447.0 (c 0.10, EtOH),
while the second eluted enantiomer afforded an experimental
[R]22 ) -439.7 (c 0.10, EtOH).
D
The quantitative CD spectra of each enantiomer were
measured in ethanol (Figure 2). The CD curves of each
enantiomer are bisignate, and this experimental evidence
indicates a coupling between chromophores.11 However, the
presence of several chromophores in compound 5 and their
reciprocal interactions render the CD spectrum quite complex
and unhandy to be related to the absolute configuration.
To assign the absolute configuration to the enantiomers, we
made several attempts to get crystals suitable for single crystal
X-ray analysis. Since this approach failed, we took into account
the methodology based on the comparison of the experimental
and the calculated optical specific rotation. Examples of
assignment of the absolute configuration in conformationally
flexible molecules have recently appeared in the literature.12.
Pharmacology. The anticonvulsant activity of derivatives
(()-5, (5R)-(+)-5 and (5S)-(-)-5 against audiogenic seizures
was evaluated 30 min after intraperitoneal administration to
DBA/2 mice, a strain genetically susceptible to sound-induced
seizures. This test has been considered an excellent animal
model for generalized epilepsy and for screening new anticon-
vulsant drugs.16 The results are compared with those previously
reported for derivative 4 and reference compound 1 (Table 3).6,7
As shown in Table 3, compound (()-5 possesses anticon-
vulsant properties higher than those of prototype 1 and

578 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Zappala` et al.
Figure 5. Superimposition of (4R)-(+)-3 (C atoms are colored in white)
and (5S)-(-)-5 (C atoms are colored in green).
the same mode of action. Therefore, compound (()-5 and its
enantiomers (5R)-(+)-5 and (5S)-(-)-5 were examined for their
ability to displace [3H]CP-526,427 from the noncompetitive
quinoxaline/benzodiazepine binding site of the AMPA receptor
complex. In this assay, the quinazolinone CP-465,022 was used
as a positive control (IC50 ) 52 nM). Compound (()-5 and
(5R)-(+)-5 showed IC50 > 10 µM, whereas enantiomer (5S)-
(-)-5 showed an IC50 ) 3.27 µM, a value considerably better
than that reported8 for compound 4 (IC50 ) 32 µM) and 1 (IC50
) 12.6 µM).
Figure 4. The potential energy scan results. (A) Conformer (5R)-5A,
energy (in hartrees) versus dihedral angle C4a-C5-C1′-C2′. (B)
Conformer (5R)-5B, energy (in hartrees) versus dihedral angle C4a-
C5-C1′-C2′.
Table 2. Calculated and Experimental Specific Rotation of the (5R)-5
Enantiomer
Furthermore, compound (()-5 and its enantiomers (5R)-(+)-5
and (5S)-(-)-5 were tested for their ability to inhibit the kainate-
induced increase of the [Ca2+]i in a primary culture of rat
cerebellar granule cells (CGC) which express AMPA recep-
tors;17 1 was used as the control. The results of the CGC test
confirmed the data of the in vivo assay. Compound (()-5 at 32
µM produced a 94% inhibition of the calcium influx; its IC50 is
12 µM, equal to that of derivative 4. Such an antagonistic
activity resides entirely in its enantiomer (5S)-(-)-5 (IC50 ) 6
µM), whereas (5R)-(+)-5, up to 32 µM, did not evidence any
inhibition of the kainate-induced calcium influx. This outcome
is noteworthy since the configuration of the eutomer is opposite
to that of (4R)-(+)-3. Since the noncompetitive binding site of
the AMPA receptors, up to now, has not been characterized by
X-ray analysis, we performed a superimposition of (4R)-(+)-3
with the most populated conformation of (S)-(-)-5. This
experiment reveals an excellent overlapping of the common
functional groups as shown in Figure 5.
population
%
Erel
(kcal/ mol)
conformer
[R]D
A
B
91
9
0.00
1.38
+794.3
-746.3
Population Weighted Specific Rotation
[R]D
+655.6
Observed Optical Specific Rotation
isomer
first-eluted
second-elut ed
[R]D
+447.0
-439.7
Table 3. Anticonvulsant Activity of Compounds 1, 4, (()-5, (5R)-(+)-5
and (5S)-(-)-5 against Audiogenic Seizures in DBA/2 Mice and TD50
Values on Locomotion Assessed by Rotarod Testa
ED50, µmol/kg
clonic phase tonic phase
PIb
TD50, µmol/kg
TD50/
compds
locomotor deficit ED50
The binding affinity of compound (()-5 at the AMPA and
Gly/NMDA receptors was also evaluated by measuring its
ability to displace [3H]AMPA and [3H]glycine, respectively,
from rat cerebral cortex synaptic membranes. To assess the
functional antagonism at the NMDA receptor-ion channel
complex, we tested derivative (()-5 for its ability to inhibit the
binding of the channel blocking agent [3H]-(+)-MK-801 in rat
cortical membranes incubated with 10 µM glutamate and 0.1
µM glycine.18 The results showed that compound (()-5,
similarly to 1,8 is devoid of any affinity (IC50 > 100 µM) for
both the AMPA recognition site and Gly/NMDA receptor site,
whereas it possesses a measurable binding affinity (33% binding
inhibition at 100 µM) for the MK-801 binding site.
In summary, the enantiomers of 3,5-dihydro-5-methyl-7,8-
methylenedioxy-4H-2,3-benzodiazepin-4-one (()-5 have been
obtained by chiral HPLC, and their absolute configuration has
been assigned by a comparison of the experimental and the
calculated optical specific rotation. The pharmacological assays
put in evidence an enantioselective interaction of the enantiomer
1
4
35.8 (24.4-52.4) 25.3 (16.0-40.0)
15.4 (10.1-23.5) 10.9 (4.60-24.6)
20.2 (16.7-24.4) 8.30 (13.6-42.7)
76.1 (47.5-122)
99.1 (72.4-135)
48.5 (34.6-67.9)
2.1
6.3
2.4
>1.1
3.1
(()-5
(5R)-(+)-5 88.4 (58.0-134) 7.36 (3.3-16.6) >100
(5S)-(-)-5 11.0 (5.79-21.1) 1.74 (0.87-3.48) 34.2 (25.3-46.3)
a All compounds were given ip (at doses spanning the range 3.3-200
µmol/kg) 30 min before auditory stimulation. All data were calculated
according to the method of Litchfield and Wilcoxon;29 95% confidence
limits are given in parentheses. At least 32 animals were used to calculate
each ED50 and TD50 value. b PI, protective index, represents the ratio
between TD50 and ED50 (from the clonic phase of the audiogenic seizures).
comparable to those of its C-5 des-methylated analogue 4.
Noteworthy, (5S)-(-)-5 was approximately twice as potent as
racemic (()-5, whereas (5R)-(+)-5 was almost inactive (ED50
) 11.0 µmol/kg for (5S)-(-)-5 vs 88.4 µmol/kg for (5R)-(+)-
5, in the clonic phase).
Given that our lead compounds display their anticovulsant
activity through the inactivation of AMPA receptors,8 it was of
utmost importance to ascertain if the new derivatives showed

Synthesis of a 2,3-Benzodiazepine DeriVatiVe
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 579
methyl-6,7-methylenedioxy-1-(4-nitrophenyl)-2H-isoquinolin-3-
one (11). A solution of compound 9 (387 mg, 1.08 mmol) and
hydrazine hydrate (0.058 mL, 1.19 mmol) in n-butanol (40 mL)
was heated at reflux. After 20 h, the expected compound 10 was
collected by hot filtration and recrystallized from methanol: mp
> 300 °C (75 mg, 20%): Rf ) 0.72 (EtOAc/cyclohexane 60/40).
Anal. (C17H13N3O5) C, H, N. By cooling, the remaining butanol
solution afforded compound 11 which was filtered off and
recrystallized from methanol: mp > 300 °C (67.5 mg, 18%); Rf )
0.10 (EtOAc/cyclohexane, 60/40). Anal. (C17H13N3O5) C, H, N.
Synthesis of 1-(4-Aminophenyl)-5-methyl-7,8-methylenedioxy-
3,5-dihydro-4H-2,3-benzodia zepin-4-one (()-5. A suspension of
10 (75 mg, 0.22 mmol) and Raney-Ni (45 mg) in EtOH (30 mL)
was stirred with ammonium formate (250 mg, 4.10 mmol). The
mixture was refluxed for 2 h and then filtered through Celite. The
solvent 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 a silica gel column chromatography eluting with EtOAc/
cyclohexane: mp 142-144 °C (17 mg, 25%) (lit.21 2.75% global
yield starting from 9); Rf ) 0.74 (EtOAc). Anal. (C17H15N3O3) C,
H, N.
(5S)-(-)-5 with the noncompetitive binding site of the AMPA
receptors. This result has been rationalized through the overlap-
ping of its most populated conformation with the structure of
(4R)-(+)-3.
Experimental Section
Lipase A from Candida Antarctica, papain from Carica papaya,
acylase I from Aspergillus melleus, lipase PS from Pseudomonas
cepacia, and R-chymotripsin were purchased from Fluka. Subtilisin
Carlsberg (Proleather) was obtained from Amano Pharmaceuticals
Co. Lipase B from Candida Antarctica was bought from Roche
Diagnostics (CHIRAZYME L-2). Carboxylesterase NP (Biofine
Esterase) was purchased from International Bio-Synthetics (Rijswick,
The Netherlands). The enzyme activity is expressed in ECU/g using
(S)-naproxen methyl ester as the substrate. One esterase cleavage
unit (ECU) is defined as the quantity of enzyme that will form 1
µmol of (S)-naproxen acid per liter per minute at pH 7.5 and 25
°C. 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 analyzer for C, H, and N, and the results
are within (0.4% of the theoretical values. Merck silica gel 60
F
254 plates were used for analytical TLC; column chromatography
was performed on Merck silica gel 60 (70-230 mesh). 1H and 13
C
Preparative Enzymatic Hydrolysis of Methyl 2-(3,4-Methyl-
enedioxyphenyl)propionate [(()-8]. To a suspension of (()-8 (1.5
g, 7.21 mmol) in 0.1 M phosphate buffer pH 7.0 (80 mL) was added
carboxylesterase NP (2 mL; 200 U/mL), and the mixture was
vigorously stirred at room temperature for 8 h. The progress of the
reaction was monitored by HPLC (column: Chirlacel OD-H;
eluant: petroleum ether/2-propanol 100:0.5; flow rate: 0.5 mL/
min; λ ) 254 nm). The reaction was stopped at 40% conversion to
yield acid (+)-12 (e.e. 83.2%) and residual ester (-)-8 (e.e. 57.2%).
Solid NaHCO3 was added, the reaction mixture was extracted with
ethyl acetate, and the pooled organic layers were evaporated to
recover the residual ester (-)-8 (890 mg, 59%). The aqueous phase
was made acid with 2 N HCl, and the monoacid (+)-12 (540 mg,
39%) was obtained after extraction with ethyl acetate, drying over
NMR spectra were recorded in CDCl3 or DMSO-d6 by means of a
Varian Gemini 300 spectrometer (see Supporting Information). The
HPLC system consisted of a PU 980 pump, a LG-1580-02 low
pressure mixer, and a DG-1580-54 in line degasser, all from Jasco,
with a Rheodyne injection valve equipped with 20 or 100 µL sample
loops, a Varian DU-50 spectrophotometer, and a Varian 4400
integrator or Houston Omniscribe recorder for fraction collecting.
The polysaccharide derived columns (250 mm × 4.6 mm) were
Chiralcel OD-H (cellulose tris[3,5-dimethylphenylcarbamate]) and
Chiralpak AS-H (amylose tris[(S)-R-methylbenzylcarbamate]) coated
on 5 µm silica gel from Daicel. Column void volume (t0) was
measured by injection of tri-tert-butylbenzene as a nonretained
sample.19 The HPLC parameters (k, R, and Rs) were those typically
employed.20 CD spectra were recorded in ethanol on a Jasco 810
spectropolarimeter using a 1 mm cell and scanning five times from
400 to 200 nm at 50 nm/min. Optical rotations were measured in
ethanol with a Jasco DIP-730 digital polarimeter using a 10 cm
microcell.
anhydrous Na2SO4, and evaporation of the solvent. (-)-8: [R]20
:
D
-26.1 (c 1.03, CHCl3). Colorless oil. Rf ) 0.78 (CH2Cl2/MeOH,
95:5). HPLC retention time: (-)-8, 29.56 min (78.6%); (+)-8,
33.46 min (21.4%) (column: Chiralcel OD-H; eluant: petroleum
ether/ 2-propanol 100:0.5; flow rate: 0.5 mL/min; λ ) 254 nm);
e.e. 57.2%. (+)-12: [R]20D: +25.5 (c 1.07, CHCl3). Colorless oil.
Rf ) 0.27 (CH2Cl2/MeOH, 95:5). HPLC retention time: (+)-12,
81.02 min (91.6%); (-)-12, 63.96 min (8.4%) (column: Chiralcel
OD-H; eluant: petroleum ether/2-propanol 100:0.5; flow rate: 0.5
mL/min; λ ) 254 nm); e.e. 83.2%. Anal. (C10H10O4) C, H.
Preparative HPLC Separation of (+)-5 and (-)-5 and Their
Chiroptical Measurements. The resolution of (()-5 was carried
out using a Chiralpak AS-H column (amylose tris[(S)-R-metilben-
zylcarbamate]). Eluent: n-hexane/ethanol 1:1. Flow rate at 1 mL/
min; t0 ) 2.90; UV detector λ ) 240 nm. The isolation was
accomplished by 50 µL repeated injections (0.2-0.3 mg) of a
solution of (()-5 (68 mg in 10 mL n-hexane/ethanol 1:1). Collection
of the eluates corresponding to the two chromatographic peaks gave,
after filtration and evaporation, 17 mg of each enantiomer. The
analytical control performed on the first collected fraction gave an
enantiomeric excess of 100% whereas the e.e. of the second fraction
amounted to 97.7%. Specific rotation of the first-eluted enantiomer
Synthesis of Methyl 2-(3,4-Methylenedioxyphenyl)propionate
(8). To a stirred solution of 3,4-methylenedioxyphenylacetic acid
methyl ester (7) (730 mg, 3.76 mmol) in anhydrous THF (40 mL)
at -70 °C was added potassium hydride (166 mg, 4.14 mmol).
After 30 min was added methyl iodide (587 mg, 4.14 mmol),
keeping the temperature at -70 °C. The cooling bath was removed,
and the reaction mixture was stirred for further 16 h, then diluted
with 50% acetic acid, poured into water, and extracted with diethyl
ether (2 × 150 mL). The combined organic layers were washed
with a saturated solution of Na2CO3 and dried over Na2SO4. The
solvent was then removed to give compound 8 (681 mg, 86.8%)
(lit.21 63%) as a colorless oil: Rf ) 0.59 (diethyl ether/cyclohexane
40/60). Anal. (C11H12O2) C, H.
Synthesis of Methyl 2-[2-(4-Nitrobenzoyl)-4,5-methylene-
dioxy-phenyl]propionate (9). 4-Nitrobenzoic acid (711 mg, 4.25
mmol) and phosphorus pentoxide (2.0 g) were added to a stirred
1,2-dichloroethane solution (50 mL) of 8 (681 mg, 3.27 mmol).
The mixture was further stirred at room temperature overnight, then
water (30 mL) was cautiously added, and the mixture was extracted
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 organic phase was dried (Na2SO4)
and the solvent removed under reduced pressure to yield crude 9
which was purified by a silica gel column chromatography using
diethyl ether/light petroleum (40/60) as eluant: mp 118-120 °C
(387 mg, 33%) (lit.21 12.3%); Rf ) 0.35 (diethyl ether/light
petroleum 40/60). Anal. (C18H15NO7) C, H, N.
was [R]22 ) +447.0 (c 0.10, EtOH), while the second eluted
D
enantiomer afforded an experimental [R]22 ) - 439.7 (c 0.10,
D
EtOH). Due to the limited amount of the individual enantiomers
of 5, first ethanolic solutions of them (about 34 × 10-4 M) were
made for optical rotation measurements, then these solutions were
diluted 1:10 with ethanol, and CD spectra were measured. After
chiroptical measurements, the solutions of the individual enanti-
omers were taken to dryness in a drying pistol in a vacuum and in
the presence of P2O5 as desiccant and the powder residues used
for pharmacological tests.
Synthesis of 3,5-Dihydro-5-methyl-7,8-methylenedioxy-1-(4-
nitrophenyl)-4H-2,3-benzodiazepin-4-one (10) and 2-Amino-4-
Assignment of the Absolute Configurations. Molecular struc-
tures were built by GView implemented in the Gaussian0313

580 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Zappala` et al.
Windows package. Both 5-methyl-benzodiazepine ring conformers,
following the preliminary optimization by semiempirical/PM322
methods, were submitted to the 360° PES analysis (step size )
30°) at B3LYP/6-31G(d) level considering the torsion angle among
the 4-aminophenyl group and the benzodiazepine ring (highlighted
in Figure 2A). The lowest potential energy was found at a torsion
angle value of -150° (or +30° due to symmetry of the molecules)
and +150° for 5A and 5B, respectively. These values of the torsion
angle were then used for a re-optimization in vacuo of the entire
structures (R)-5A and (R)-5B. Furthermore, a single point calcula-
tion, using the PCM ethanol solvent model, permitted us to estimate
the conformers population in the experimental conditions. Theoreti-
cal values of the [R]D were achieved specifying appropriate keyword
(polar and cphf) into the Gaussian input.
Audiogenic Seizures Test in DBA/2 Mice.23 DBA/2 mice (8-
12 g, 22-25 days old) were purchased from Charles River (Calco,
Como, Italy). Groups of 10 mice of either sex were randomly
assigned to controls and drug-treatment groups and 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 cm),
and 60 s were allowed for habituation and assessment of locomotor
activity. Auditory stimulation (12-16 kHz, 109 dB) was applied
for 60 s or until tonic extension occurred and induced a sequential
seizure response in control DBA/2 mice, consisting of an early wild
running phase, followed by 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 seizures.24
[3H]CP-526,427 Binding.17 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 nuclear 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 protein/mL
with 50 mM Tris‚HCl, pH 7.4. Scatchard analyses were performed
by incubating different concentrations of radioligand with mem-
branes. For competition assays, compounds were added at various
concentrations followed by 3 nM [3H]CP-526,427 (specific activity,
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 cell harvester (Brandel Research and
Development Laboratories, Gaithersburg, MD). Filters were washed
for 10 s with ice-cold Tris‚HCl buffer, and the radioactivity trapped
on the filter was quantified by liquid scintillation counting.
Nonspecific binding for [3H]CP-526,427 was determined in parallel
incubations containing 10 µM unlabeled CP-526,427 or CP-465,-
022. Specific binding was defined as total binding minus nonspecific
binding.
of the lysate were measured with a TopCount microtiter scintillation
counter (Packard Instrument Co., Downers Grove, IL).
Measurement of [Ca2+]i.17 Neurons in 96-well, black/clear, poly-
D-lysine-coated tissue culture plates were rinsed once with BSS
then incubated for 1 h in BSS containing 4 µM Fluo-4/AM
(Molecular Probes, Inc., Eugene, OR). Fluo-4/AM was prepared
immediately 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 temperature 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 ap-
proximately 6 min, then stimulated with 32 µM 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.
[3H]AMPA Binding. Affinity for the competitive AMPA
receptor binding site was determined with 5 nM [3H]AMPA as
previously described.26
[3H]Glycine Binding. These experiments were carried out in
rat cortical synaptic membrane preparations with 8 nM [3H]glycine
as ligand, according to the method described.27
[3H]-(+)-MK-801 Binding. [3H]-(+)-MK-801 ((+)-5-methyl-
10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate)
binding assays were carried out according to Yoneda et al.28 with
slight modifications. Preparations of frozen rat cortical membranes
were resuspended (0.5 mg protein/mL) in ice-cold 5 mM Tris-HCl
buffer, pH 7.4, containing 0.08% v/v Triton X-100 and stirred for
10 min at 0-2 °C. They were then collected by centrifugation
(48 000g for 10 min) and submitted to four additional resuspension
and centrifugation cycles before finally being resuspended in the
appropriate volume of buffer (0.2-0.3 mg protein/tube) for the
binding assay. The assay incubations were carried out at room
temperature for 120 min with 2.5 nM [3H]-(+)-MK-801 (22.5 Ci/
mmol), 10 mM glutamic acid, and 0.1 mM glycine in the presence
and absence of the test compound in a total volume of 0.5 mL.
Bound radioactivity was separated by filtration through GF/C filters
presoaked in 0.05% polyethylenimmine and washed with ice-cold
buffer (3 × 5 mL). Nonspecific binding was determined in the
presence of 100 mM phencyclidine hydrochloride.
Statistical Analysis. The ED50 values of each phase of the
audiogenic seizure were determined for each dose of compound
administered, and dose-response curves were fitted using a
computer program by the method of Litchfield and Wilcoxon.29
The median toxic dose (TD50) values were estimated using the
method of Litchfield and Wilcoxon.29
Acknowledgment. This work was financially supported by
the Ministero dell’Istruzione, dell’Universita` e della Ricerca
Scientifica e Tecnologica (MIUR - COFIN2004) - Rome. The
authors wish to thank Anne Schmidt, Pfizer Inc., for excellent
technical assistance.
Supporting Information Available: Analytical and spectral
data of all the synthesized compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Primary Cultures of Rat Cerebellar Granule Neurons.
Primary cultures of rat cerebellar granule neurons were prepared
as described previously.25
45Ca2+ Uptake.17 Neurons in poly-D-lysine-coated 96-well plates
were preincubated for 30 min with different concentrations of
compounds in balanced salt solution (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 µM kainate or NMDA in BSS containing 10
µM glycine, 0.5 mM dithiothreitol, and 0.5 µCi 45Ca2+ (final specific
activity, 2.78 µCi/µmol) in a volume of 100 µL/well. After 10 min,
the neurons were then rapidly washed five times with 200 µL/well
of ice-cold BSS containing 5 mM EGTA. Neurons were then lysed
in 30 µL/well of 0.6% Triton X-100, and radioactivity in aliquots
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