Indeno[1,2-b]pyrazin-2,3-diones: A new class of antagonists at the glycine site of the NMDA receptor with potent in vivo activity

Article abstract of DOI:10.1021/jm990957g

Indeno[1,2-b]pyrazin-2,3-diones have been identified as a novel series of potent ligands on the glycine site of the NMDA receptor. To improve their in vivo activities, an acetic acid-type side chain was introduced to the 5- position, giving water-soluble

Full text of DOI:10.1021/jm990957g

J . Med. Chem. 2000, 43, 2371-2381
2371
In d en o[1,2-b]p yr a zin -2,3-d ion es: A New Cla ss of An ta gon ists a t th e Glycin e Site
of th e NMDA Recep tor w ith P oten t in Vivo Activity
Patrick J imonet,* Yves Ribeill,† Georg Andrees Bohme, Alain Boireau, Michel Cheve´, Dominique Damour,
Adam Doble, Arielle Genevois-Borella, Fre´de´ric Herman, Assunta Imperato, Sylvain Le Guern, Franco Manfre´,
J eremy Pratt, J ohn C. R. Randle,‡ J ean-Marie Stutzmann, and Serge Mignani
Department of Medicinal Chemistry and CNS Program, Aventis Pharma S.A., Centre de Recherche de Vitry-Alfortville,
13 Quai J ules Guesde, BP 14, 94403 Vitry-sur-Seine Cedex, France
Received November 22, 1999
Indeno[1,2-b]pyrazin-2,3-diones have been identified as a novel series of potent ligands on the
glycine site of the NMDA receptor. To improve their in vivo activities, an acetic acid-type side
chain was introduced to the 5-position, giving water-soluble compounds when formulated as
the sodium salt (>10 mg/mL). Introduction of a chlorine atom in the 8-position led to a dramatic
improvement of anticonvulsant activity and this was surprising since this change did not
improve binding affinity. A plausible explanation is a reduced recognition by a Na+,K+-ATPase
active transport system responsible for the excretion of these compounds from the brain and
kidney. This promising new chemical series led to the optically active isomer (-)-10i (RPR
118723), a glycine/NMDA antagonist with nanomolar binding affinity and in vivo activity in
animal model of convulsions and electrophysiology at doses in the range of 2-3 mg/kg following
iv administration.
It is now well-established that excessive stimulation
of ionotropic glutamate receptors (NMDA and AMPA
receptors) is implicated in the neuronal death that
occurs in various neurological disorders including ce-
rebral ischemia and neurotrauma.1 Competitive and
noncompetitive NMDA antagonists acting at the NMDA
subtype of the glutamate receptor have been proposed
as potential neuroprotective agents in humans, since
they have demonstrated effective neuroprotection in
animal models.2 However, both competitive antagonists
and channel blockers produce side effects that may limit
their utility as therapeutic agents.3 The discovery that
glycine acts as a coagonist at a specific site of the NMDA
receptor-channel complex4 has led to an intensive search
for antagonists acting at this modulatory site; such
compounds represent an alternative way to antagonize
NMDA neurotransmission with potentially fewer sec-
F igu r e 1. NMDA antagonists acting on the glycine site
ondary effects.5 To date, few selective glycine antago-
entered in clinical trials.
nists have been shown to possess potent in vivo activity
and only three of these (Figure 1) have entered clinical
been removed might prove to be more efficient in
penetrating the blood-brain barrier by analogy with
quinolin-2-ones 8 vs kynurenic acid derivatives 7.5d,9a
Second, we were seeking a new heterocycle allowing
more versatile chemical derivatization. We designed
pyrazin-2,3-diones bearing a phenyl group in position
5 (5) as a substitute for the fused benzo ring of
quinoxalines. Formal cyclization between the two rings
yielded a pseudoplanar heterocycle in the indeno[1,2-
b]pyrazine-2,3-diones 6. Such a type of introduction of
a saturated benzo-fused moiety has already been de-
scribed but in a different position of the molecule (e.g.
SM-18400 9).9b Preparation and pharmacological evalu-
ation of the parent compound (6a ) demonstrated en-
couraging antagonist activity (IC50 ) 350 nM) at the
glycine-NMDA binding site.10 Introduction of a chloro
substituent at position C-8 increased affinity greater
than 10-fold (6b, IC50 ) 25 nM), an observation well-
trials.5b,6
Following our previous identification of the ben-
zothiadiazine RPR 104632 (4) as a potent glycine/NMDA
antagonist in vitro but with limited in vivo activity,7
research was focused on novel chemical families, par-
ticularly on the indeno[1,2-b]pyrazin-2,3-diones such as
6a and 6b (Figure 2).8 Our initial design was driven by
two concepts. First, we wished to capitalize on an
apparent structural similarity between the dione system
of quinoxalin-2,3-diones (e.g. ACEA 1021, 1) and the
carboxylic acid of the benzothiadiazine 4; we hypoth-
esized that compounds in which the carboxylic acid had
* Corresponding author. E-mail: patrick.jimonet@aventis.com.
† Present address: Aventis Crop Science, Research Triangle Park,
NC 27709.
‡ Present address: Vertex Pharmaceuticals Incorporated, Cam-
bridge, MA 02139-4242.
10.1021/jm990957g CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/19/2000

2372 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
J imonet et al.
substitution in position 5 of our original series of
indeno[1,2-b]pyrazin-2,3-diones with a general formula
10 (Figure 2).
Ch em istr y
Compounds 10a -j (Table 1) were obtained in 10 steps
from appropriate aryl ketones 11 following the chemical
pathway shown in Scheme 1. Knoevenagel condensation
of these ketones with ethyl cyanoacetate followed by
Michael addition of cyanoacetamide to the resulting
R-cyanocinnamates 12, obtained as an isomeric mixture,
led to cyclic imides 13. Sulfuric acid hydrolysis and
decarboxylation of 13 gave diacids 14 which were
cyclized under standard Friedel-Craft conditions to give
indanones 15. The R-amino derivatives 18 were pre-
pared by hydrogenolysis of the oximes 17 which were
obtained from reaction between the keto esters 16 and
tert-butyl nitrite. Then, the pyrazino ring was con-
structed in the final steps by reacting the amino ketones
18 with ethyl oxalyl chloride followed by cyclization in
the presence of excess ammonium acetate in refluxing
acetic acid. Final acidic hydrolysis of the ethyl esters
20 using 8 N HCl gave the desired indeno[1,2-b]pyra-
zine-2,3-diones 10a -j.
F igu r e 2. Bicyclic and tricyclic NMDA antagonists acting on
the glycine site.
correlated with the increased affinity observed previ-
ously in other heterocyclic families upon related chloro
substitutions.7 Unfortunately, when examined in our
primary in vivo screen, the maximum electroshock-
induced seizure (MES) test in mice,11 compound 6b
showed very low activity (ED50 > 80 mg/kg ip). Because
of the poor aqueous solubility of 6b (<1 mg/mL) and a
potential for binding to plasma proteins12 (no experi-
ment done), we reexamined incorporation of a carboxylic
acid group, but in a different position which could be
tolerated by the binding site. Despite a negative impact
on physicochemical parameters (e.g. log D), such a
strategy to increase solubility and potency of glycine/
NMDA antagonists was already reported successfully
in the quinoxalin-2-one and quinoxalinedione series.5d,9b
These results encouraged us to pursue acetic acid
Resu lts a n d Discu ssion
The affinities of compounds 1, 4, and 10a -j for the
glycine/NMDA and 2-amino-3-(3-hydroxy-5-methylisox-
azol-4-yl)propionic acid (AMPA) receptors were evalu-
ated using [3H]dichlorokynurenate ([3H]DCKA) and
[3H]AMPA displacements from rat cortical mem-
branes.10a,b In vivo activities of these compounds were
measured in the mouse maximal electroshock convul-
sion test;11 the respective IC50 and ED50 values in these
tests are reported in Table 1.
On the basis of the DCKA binding data, unexpected
structure-activity relationships were observed: intro-
duction of a chlorine atom to position 8, which increased
Ta ble 1. Binding Affinity and Anticonvulsant Activity of Indeno[1,2-b]pyrazin-2,3-diones 10a -j
(IC50, nM)
MES mice
R1 , R2
R3
[3H]DCKAa
[3H]AMPAb
4600
(ED50, mg/kg ip)c
10a
H
CH3
21 ( 1
(n ) 3)
79
65
with probenecid:
423
187
2.7
80
>80
>80
>80
23
10b
10c
10d
10e
10f
10g
10h
10i
6-Cl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
6-Cl, 8-Cl
7-Cl, 8-Cl
8-Cl, 9-Cl
8-CH3
8-F
24
140
2550
12
15
4300
28
1800
>10000
4500
1280
>80
n.d.
6.8
8-OCF3
8-Cl
>10000
2100
with probenecid:
1.8
10j
1
4
8-Cl
ACEA 1021
RPR 104632
CH2CH2CH3
21
4
8.3 ( 0.3
(n ) 6)
7000
766
>10000
9.6
22.5
>80
a
The accuracy of the assay is reflected by SEM determined for compounds tested at least in three experiments run in duplicate
(compounds 4 and 10a ). Other compounds were tested once in duplicate. Compounds tested once in duplicate. c Compounds tested in
b
one experiment only. The variability in this test could be estimated to be around 12% (see Experimental Section).

Indeno[1,2-b]pyrazin-2,3-diones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2373
Sch em e 1. Synthesis of Compounds 10a -j
the affinity of the “lead compound” 6b did not show any
significant effect on the binding affinity (10i vs 10a ).
Likewise, introduction of other substituents such as a
methyl group (10f) or a fluorine atom (10g) in position
8 or chlorine atoms in positions 6 and 8 (10c) led to
compounds only as active as the unsubstituted dione
10a . On the other hand, and analogous to observations
for the bicyclic series of glycine-NMDA antagonists,13
introduction of substituents in positions 7 or 9 gave less
active derivatives (10d and 10e). For the R3 substituent,
lengthening the alkyl chain (n-propyl instead of methyl)
did not change affinity for the binding site, indicating
room for hydrophobic substituents on the side opposite
to the carboxylic group. Affinities for the other ionotropic
glutamate receptor (AMPA) were only found in the
micromolar range or above, except for the two deriva-
tives substituted in position 6 (10b,c).
In vivo studies of these compounds showed even more
interesting results: the weak anticonvulsant activity
observed with the unsubstituted carboxylic acid 10a
(ED50 ) 65 mg/kg ip) was encouraging in comparison
with the lack of activity of the parent compounds 6a
and 6b. More dramatic was the large increase of
anticonvulsant effect shown by the 8-chloro derivative
10i (ED50 ) 6.8 mg/kg ip). It is interesting to notice that
the other derivatives are inactive, except the methyl
analogue 10f and the R3-modified compound 10j. These
results could not be explained by an interaction with
the AMPA receptor, since no correlation was observed
between the affinity for this receptor and the anticon-
vulsant activity. The in vivo activity of several excitatory
amino acid antagonists has been shown to be increased
by probenecid (p-(dipropylsulfamoyl)benzoic acid), a
lipid-soluble competitive inhibitor of transport systems
coupled to Na+,K+-ATPase and organic ion transport.
This is interpreted to be an indication that their
excretion by the kidneys and perhaps brain penetration
are affected by organic acid transport systems.14 The
anticonvulsant potency of compound 10a was consider-
ably increased following pretreatment with probenecid
(200 mg/kg ip) with ED50 reduced from 65 to 2.7 mg/kg
ip. A smaller effect was observed for compound 10i.
Thus 10a and 10i have similar DCKA binding affinities
(21 and 28 nM, respectively) and, in the presence of
probenecid, similar in vivo anticonvulsant activities (2.7
and 1.8 mg/kg ip, respectively). These results suggest
that a Na+,K+-ATPase active transport system may be
responsible for the excretion of these compounds from
the central nervous system and kidneys. It appears that
10i is poorly recognized by this transport system and
thus is able to exert a powerful anticonvulsant effect.
Com p ou n d (-)-10i (RP R 118723): P r ep a r a tion
a n d Biologica l Activities. Since compound 10i exists
as a racemate, we wished to examine the activities of
the two enantiomers. The enantiomers of compound 10i
were prepared in optically pure form by HPLC separa-
tion of the enantiomers of the ethyl ester precursor
(column packed with Chiralcel OD as stationary phase
and ethanol used as eluent) followed by 6 N HCl
hydrolysis. The enantiomeric excess for both enanti-
omers (-)-10i and (+)-10i (>99.5%) was evaluated by
HPLC using the same chiral phase and 5/95 ethanol/
heptane containing 0.05% trifluorocaetic acid as eluent.
As shown in Table 2, the levorotatory isomer (-)-10i
(RPR 118723) is about 8 times more potent as a ligand
of the glycine/NMDA binding site than the dextrorota-
tory isomer (+)-10i (5 ( 1 nM vs 44 ( 9 nM). More
strikingly, the dextrorotatory enantiomer showed no
significant anticonvulsant activity at 80 mg/kg, whereas
the former displayed a very potent protective effect

2374 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
J imonet et al.
Ta ble 2. Binding Affinity and Anticonvulsant Activity of Racemic 10i and Enantiomers (-)-10i and (+)-10i
(IC50, nM)
MES mice (ED50, mg/kg)c
[R]20 (c ) 1, DMF)
[3H]DCKAa
[3H]AMPAb
ip
iv
D
10i
28
2100
6.8
nt
(-)-10i
RPR 118723
(+)-10i
-72.5 ( 1.1
+73.2 ( 1.1
5 ( 1
(n ) 4)
44 ( 9
(n ) 3)
10000
4.5 ( 0.5
(n ) 8)
>80
2.0
1000
nt
a
The accuracy of the assay is reflected by SEM determined for compounds tested at least in three experiments run in duplicate. Other
compounds were tested once in duplicate. Compounds tested once in duplicate. c The variability in this test could be estimated to be
b
around 12% (see Experimental Section).
F igu r e 3. Effect of (-)-10i on NMDA-evoked functional
responses in neurons in culture. Traces of NMDA (50 µM)-
evoked current (1) and inhibition after a 5 min preincubation
with (-)-10i at 10 nM (2) and after 3 min (3) and 20 min (4)
of wash-out. Note the reversible inhibition of NMDA-receptor
mediated responses by (-)-10i.
F igu r e 4. Brain penetration of (-)-10i as measured by effect
on long-term potentiation in vivo. Time course of change in
following either ip or iv administration (ED50 ) 4.5 and
2.0 mg/kg, respectively). Moreover, (-)-10i is a very
weak ligand for the AMPA receptor (IC50 ) 10 µM), as
well as for the glutamate and TCP binding sites of the
NMDA receptor (IC50 ) 5.2 and 25 µM, respectively).
Compound (-)-10i was therefore chosen for electro-
physiological evaluation in vitro and in vivo. Currents
activated by NMDA in rat cerebellar neurons in culture
were dose-dependently antagonized by (-)-10i with an
IC50 of 1.9 nM (Figure 3). To evaluate whether (-)-10i
penetrates the blood-brain barrier in concentrations
sufficient to inhibit NMDA receptor activation in vivo,
we examined its effect on long-term potentiation (LTP)
in the brain of anesthetized rat. LTP is a synaptic
plasticity phenomenon induced by electrical stimulation
in the hippocampus that is dependent on local NMDA
receptor activation. Intravenous injections of (-)-10i
dose-dependently blocked LTP over the 0.3, 3 and 30
mg/kg dose-range (Figure 4). This effect was significant
at 3 mg/kg (Figure 5), a value similar to that at which
anticonvulsant activity was observed in the MES test
in mice.
the mean ((SEM) normalized amplitude of the epsps recorded
in the CA1 area of the hippocampus in intact, anesthetized
rats following three sets of high-frequency stimulation (3 ×
HFS). O ) Vehicle-treated controls (saline); b ) (-)-10i, 0.3
mg/kg iv; (1 ) (-)-10i, 3 mg/kg iv; 0 ) (-)-10i, 30 mg/kg iv.
F igu r e 5. Dose-dependency of the effect of (-)-10i on LTP
in vivo. Bars represent mean epsp size measured 60 min after
HFS. Symbols show individual data. The asterisk shows
significant difference from controls (p < 0.55, Dunnett’s
multiple comparison test).
Con clu sion
soluble glycine/NMDA antagonist with nanomolar af-
finity in vitro. Furthermore, (-)-10i has in vivo activity
in an anticonvulsant model and in pharmacodynamic
models of NMDA receptor function at doses in the range
of 2-3 mg/kg by intravenous route.
Indeno[1,2-b]pyrazin-2,3-diones have been found to be
a novel series of potent ligands on the glycine site of
the NMDA receptor. To improve their in vivo activities,
an acetic acid-type side chain was introduced to the
5-position, leading to water-soluble compounds as so-
dium salts (>10 mg/mL). Introduction of a chlorine atom
to the 8-position led to a dramatic improvement of
anticonvulsant activity without any effect on binding
affinity. A likely explanation is a reduced recognition
by a Na+,K+-ATPase active transport system respon-
sible for the excretion of these compounds from the brain
and kidney. This promising new chemical series led to
the optically active (-)-10i (RPR 118723), a water-
Exp er im en ta l Section
Ch em istr y. Gen er a l. Melting points were determined on
a Ko¨fler apparatus and are uncorrected. 1H NMR spectra were
recorded on a Bruker AC 200 (200 MHz), a Bruker AC 250
(250 MHz) or a Bruker AC 300 (300 MHz). NMR data are
reported in ppm downfield relative to external TMS (0 ppm)
as standard. IR spectra were taken on an IRTF Nicolet 510.
Elemental analyses are indicated by the symbol of the ele-
ments and the results were within (0.4% (for C, H, N) of the

Indeno[1,2-b]pyrazin-2,3-diones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2375
theoretical values unless otherwise noted. Medium-pressure
chromatographic separations were performed on silica gel
(0.04-0.063 mm supplied by Merck). All organic solutions were
dried over magnesium sulfate. Yields are not optimized.
Gen er a l P r oced u r e A. Eth yl 2-Cya n o-3-(2-ch lor op h en -
yl)-2-bu ten oa te (12a ). A mixture of 51.4 g (0.33 mol) of
2-chloroacetophenone, 36 mL (0.33 mol) of ethyl cyanoacetate,
5 g (0.066 mol) of ammonium acetate, and 15 mL of acetic acid
in 100 mL of toluene is heated to reflux overnight and the
water formed during the reaction is removed by azeotropic
distillation. After returning to room temperature, the reaction
mixture is concentrated to dryness and the residue solubilized
in 500 mL of ethyl acetate and then washed twice with 100
mL of distilled water and twice with 100 mL of saturated
aqueous sodium chloride solution, dried over magnesium
sulfate, and concentrated to dryness under reduced pressure.
The crude oil obtained is distilled under reduced pressure (1
mmHg). A 50/50 Z/E isomeric mixture of ethyl 2-cyano-3-(2-
chlorophenyl)-2-butenoate (12a ) (54 g, 66%) is thereby obtained
d6) 1.80 (s, 3H, CH3), 5.80 (s, 2H, 2 CH), 7.5-7.90 (m, 4H, ArH),
12.30 (br s, 1H, NH).
Compounds 13b-g were obtained following procedure B.
2,4-Dicya n o-3-m eth yl-3-(2,4-d ich lor op h en yl)glu ta r im -
id e (13b): white crystals (26.5 g, 19%), mp >260 °C; 1H NMR
δ (300 MHz, DMSO-d6) 1.75 (s, 3H, CH3), 5.75 (s, 2H, 2 CH),
7.60 (d, 1H, J ) 8 Hz, ArH), 7.80 (m, 2H, ArH), 12.30 (s, 1H,
NH).
2,4-Dicya n o-3-m eth yl-3-(3,4-d ich lor op h en yl)glu ta r im -
id e (13c): beige crystals (56 g, 87%), mp >260 °C; 1H NMR δ
(300 MHz, DMSO-d6) 1.70 (s, 3H, CH3), 5.40 (s, 2H, 2 CH),
7.65 (dd, 1H, J ) 8 and 2 Hz, ArH), 7.82 (d, 1H, J ) 8 Hz,
ArH), 7.98 (d, 1H, J ) 2 Hz, ArH), 12.40 (s, 1H, NH).
2,4-Dicya n o-3-m et h yl-3-(4-flu or op h en yl)glu t a r im id e
1
(13d ): yellow solid (62 g, 89%), mp 255 °C; H NMR δ (300
MHz, DMSO-d6) 1.68 (s, 3H, CH3), 5.32 (s, 2H, 2 CH), 7.40 (t,
2H, J ) 8 Hz, ArH), 7.75 (dd, 2H, J ) 8 and 4 Hz, ArH), 12.35
(s, 1H, NH).
2,4-Dicya n o-3-m et h yl-3-(4-t r iflu or om et h oxyp h en yl)-
glu ta r im id e (13e): beige crystals, (18 g, 93%), mp 225 °C;
1H NMR δ (250 MHz, DMSO-d6) 1.72 (s, 3H, CH3), 5.38 (s,
2H, 2 CH), 7.55 (d, 2H, J ) 8 Hz, ArH), 7.82 (d, 2H, J ) 8 Hz,
ArH), 12.40 (s, 1H, NH).
1
in the form of a colorless oil. H NMR δ (300 MHz, DMSO-d6)
0.95 and 1.30 (t, 3H, J ) 7 Hz, CH3), 2.50 and 2.60 (s, 3H,
CH3), 4.00 and 4.30 (q, 2H, J ) 7 Hz, OCH2), 7.25-7.65 (m,
4H, ArH).
Compounds 12b-g were obtained following procedure A.
Eth yl 2-cyan o-3-(2,4-dich lor oph en yl)-2-bu ten oate (12b):
2,4-Dicya n o-3-m et h yl-3-(4-ch lor op h en yl)glu t a r im id e
1
(13f): yellow solid (151 g, 87%), mp 261 °C; H NMR δ (300
1
pale yellow oil (122 g, 75%), 60/40 Z/E isomeric mixture; H
MHz, DMSO-d6) 1.70 (s, 3H, CH3), 5.32 (s, 2H, 2 CH), 7.65-
7.85 (m, 4H, ArH), 12.40 (s, 1H, NH).
NMR δ (300 MHz, DMSO-d6) 1.08 and 1.35 (t, 3H, J ) 7 Hz,
CH3), 2.52 and 2.64 (s, 3H, CH3), 4.10 and 4.35 (q, 2H, J ) 7
Hz, OCH2), 7.41 and 7.57 (d, 1H, J ) 8 Hz, ArH), 7.57 and
7.66 (dd, 1H, J ) 2 and 8 Hz, ArH), 7.78 and 7.89 (d, 1H, J )
2 Hz, ArH).
Eth yl 2-cyan o-3-(3,4-dich lor oph en yl)-2-bu ten oate (12c):
colorless solid (58 g, 41%), 65/35 Z/E isomeric mixture; 1H NMR
δ (250 MHz, DMSO-d6) 1.08 and 1.33 (t, 3H, J ) 7 Hz, CH3),
2.52 and 2.66 (s, 3H, CH3), 4.10 and 4.33 (q, 2H, J ) 7 Hz,
OCH2), 7.34 and 7.58 (dd, 1H, J ) 2 and 8 Hz, ArH), 7.69 and
7.90 (d, 1H, J ) 2 Hz, ArH), 7.71 and 7.82 (d, 1H, J ) 8 Hz,
ArH).
Eth yl 2-cya n o-3-(4-flu or op h en yl)-2-bu ten oa te (12d ):
yellow oil (65.4 g, 39%), 60/40 Z/E isomeric mixture; 1H NMR
δ (300 MHz, DMSO-d6) 1.08 and 1.35 (t, 3H, J ) 7 Hz, CH3),
2.52 and 2.69 (s, 3H, CH3), 4.10 and 4.35 (q, 2H, J ) 7 Hz,
OCH2), 7.20-7.80 (m, 4H, ArH).
2,4-Dicya n o-3-p r op yl-3-(4-ch lor op h en yl)glu t a r im id e
(13g): white crystals (19 g, 60%), mp 208 °C; 1H NMR δ (300
MHz, DMSO-d6) 1.05 (t, 3H, J ) 6 Hz, CH3), 1.42 (m, 2H, CH2),
2.15 (m, 2H, CH2), 5.35 (s, 2H, 2 CH), 7.40-7.70 (m, 4H, ArH),
12.40 (s, 1H, NH).
Gen er a l P r oced u r e C. 3-(2-Ch lor op h en yl)-3-m eth yl-
p en ta n ed ioic Acid (14a ). A solution of 18 g (0.063 mol) of
2,4-dicyano-3-methyl-3-(2-chlorophenyl)glutarimide (13a ) in a
mixture consisting of 75 mL of water, 75 mL of concentrated
sulfuric acid, and 50 mL of acetic acid is heated to reflux for
40 h. After returning to room temperature, the reaction
medium is poured into 500 mL of ice-cold water. The organic
phase is extracted with ethyl acetate (2 × 250 mL) and then
washed with distilled water (3 × 100 mL), dried over magne-
sium sulfate, filtered, and concentrated to dryness under
reduced pressure. The crude product is triturated in 50 mL of
water, filtered, and dried to give 13.6 g (85%) of 3-(2-
chlorophenyl)-3-methylpentanedioic acid (14a ) as white crys-
Eth yl 2-cya n o-3-(4-tr iflu or om eth oxyp h en yl)-2-bu ten -
oa te (12e): pale yellow oil (17.2 g, 35%), 65/35 Z/E isomeric
mixture; 1H NMR δ (300 MHz, DMSO-d6) 1.00 and 1.32 (t,
3H, J ) 7 Hz, CH3), 2.55 and 2.70 (s, 3H, CH3), 4.04 and 4.32
(q, 2H, J ) 7 Hz, OCH2), 7.40-7.75 (m, 4H, ArH).
Eth yl 2-cya n o-3-(4-ch lor op h en yl)-2-bu ten oa te (12f):
1
tals melting at 158 °C. H NMR δ (300 MHz, DMSO-d6) 1.70
(s, 3H, CH3), 2.82 and 3.20 (d, 2H each, J ) 16 Hz, 2CH2CO),
7.15-7.55 (m, 4H, ArH), 12.00 (br s, 2H, 2CO2H).
Compounds 14b-g were obtained following procedure C.
1
3-(2,4-Dich lor op h en yl)-3-m et h ylp en t a n ed ioic
a cid
yellow oil (153 g, 38%), 65/35 Z/E isomeric mixture; H NMR
1
(14b): brown oil (19 g, 84%); H NMR δ (250 MHz, DMSO-
d6) 1.70 (s, 3H, CH3), 2.80 and 3.25 (d, 2H each, J ) 16 Hz,
2CH2CO), 7.38 (dd, 1H, J ) 8 and 2 Hz, ArH), 7.50 (d, 1H, J
) 8 Hz, ArH), 7.52 (d, 1H, J ) 2 Hz, ArH), 12.00 (br s, 2H,
2CO2H).
δ (300 MHz, DMSO-d6) 1.00 and 1.30 (t, 3H, J ) 7 Hz, CH3),
2.55 and 2.65 (s, 3H, CH3), 4.05 and 4.30 (q, 2H, J ) 7 Hz,
OCH2), 7.30-7.60 (m, 4H, ArH).
Eth yl 2-cya n o-3-(4-ch lor op h en yl)-2-h exen oa te (12g):
yellow oil (16.2 g, 58%), 55/45 Z/E isomeric mixture: 1H NMR
δ (300 MHz, CDCl3) 0.90-1.60 (m, 8H, CH2 and 2CH3), 2.78
and 3.02 (t, 2H, J ) 7 Hz, CH2), 4.08 and 4.30 (q, 2H, J ) 7
Hz, OCH2), 7.00-7.50 (m, 4H, ArH).
Gen er a l P r oced u r e B. 2,4-Dicya n o-3-m eth yl-3-(2-
ch lor op h en yl)glu ta r im id e (13a ). Cyanoacetamide (16.8 g,
0.2 mol) is added slowly at 5 °C to an ethanolic solution of
sodium ethylate obtained by adding 4.6 g (0.2 mol) of sodium
to 250 mL of ethanol. After 15 min, a solution of 50 g (0.2 mol)
of ethyl 2-cyano-3-(2-chlorophenyl)-2-butenoate (12a ) is added
to the suspension obtained. The reaction mixture is stirred for
4 h at room temperature and is then poured into 300 mL of
distilled water. The reaction medium is cooled to 5 °C and then
acidified to pH ) 1 by adding a concentrated solution of
aqueous hydrochloric acid. The precipitate is separated by
filtration, washed with water, and dried in the air. 2,4-
Dicyano-3-methyl-3-(2-chlorophenyl)glutarimide (13a) is thereby
obtained (31 g) in the form of light brown crystals which were
recrystallized in ethanol to give 19 g (33%) of pure compound
as white crystals: mp >260 °C; 1H NMR δ (300 MHz, DMSO-
3-(3,4-Dich lor oph en yl)-3-m eth ylpen tan edioic acid (14c):
orange oil (54 g, 100%); 1H NMR δ (300 MHz, DMSO-d6) 1.50
(s, 3H, CH3), 2.72 and 2.82 (d, 2H each, J ) 16 Hz, 2CH2CO),
7.34 (dd, 1H, J ) 8 and 2 Hz, ArH), 7.50 (d, 1H, J ) 8 Hz,
ArH), 7.59 (d, 1H, J ) 2 Hz, ArH), 12.00 (br s, 2H, 2CO2H).
3-(4-F lu or op h en yl)-3-m eth ylp en ta n ed ioic a cid (14d ):
1
brown solid (34.5 g, 78%), mp 102 °C; H NMR δ (300 MHz,
DMSO-d6) 1.52 (s, 3H, CH3), 2.78 (m, 4H, 2CH2CO), 7.10 (t,
2H, J ) 8 Hz, ArH), 7.40 (dd, 2H, J ) 8 and 4 Hz, ArH), 11.70
(br s, 2H, 2CO2H).
3-(4-Tr iflu or om eth oxyp h en yl)-3-m eth ylp en ta n ed ioic
a cid (14e): crude product used without any further purifica-
tion (16 g); 1H NMR δ (300 MHz, DMSO-d6) 1.55 (s, 3H, CH3),
2.80 (m, 4H, 2CH2CO), 7.25 (d, 2H, J ) 8 Hz, ArH), 7.50 (d,
2H, J ) 8 Hz, ArH), 12.00 (s, 2H, 2CO2H).
3-(4-Ch lor op h en yl)-3-m et h ylp en t a n ed ioic a cid (14f):
1
pale yellow oil (0.6 g, 48%); H NMR δ (300 MHz, DMSO-d6)
1.52 (s, 3H, CH3), 2.77 and 3.02 (d, 2H each, J ) 16 Hz,
2CH2CO), 7.40 (m, 4H, ArH), 12.30 (s, 2H, 2CO2H).

2376 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
J imonet et al.
3-(4-Ch lor op h en yl)-3-p r op ylp en t a n ed ioic a cid (14g):
off-white crystals (9.7 g, 78%), mp 142 °C; 1H NMR δ (250
MHz, DMSO-d6) 0.78 (t, 3H, J ) 6 Hz, CH3), 1.00 (m, 2H, CH2),
1.80 (m, 2H, CH2), 2.95 (m, 4H, 2CH2CO), 7.37 (m, 4H, ArH).
Gen er a l P r oced u r e D. (7-Ch lor o-1-m eth yl-3-oxo-1-in -
d a n yl)a cetic Acid (15a ). A suspension of 13 g (0.05 mol) of
3-(2-chlorophenyl)-3-methylpentanedioic acid 14a in 50 mL of
concentrated sulfuric acid is heated to reflux for 24 h. After
returning to room temperature, the reaction medium is poured
slowly into 300 mL of ice-cold water and is then extracted twice
with 300 mL of ethyl acetate. The combined organic phases
are washed with 100 mL of a saturated aqueous solution of
sodium chloride, dried over magnesium sulfate, and concen-
trated to dryness under reduced pressure; 13 g (100%) of (7-
chloro-1-methyl-3-oxo-1-indanyl)acetic acid (15a ) is thereby
obtained in the form of a yellow oil. 1H NMR δ (300 MHz,
DMSO-d6) 1.57 (s, 3H, CH3), 2.60 and 3.03 (d, 2H, J ) 16 Hz,
CH2), 2.93 and 3.12 (d, 2H, J ) 14 Hz, CH2), 7.47 (t, 1H, J )
7 Hz, ArH), 7.60 (d, 1H, J ) 7 Hz, ArH), 7.70 (d, 1H, J ) 7
Hz, ArH), 12.10 (br s, 1H, CO2H).
Compounds 15b-h were obtained following procedure D.
5,7-Dich lor o-1-m eth yl-3-oxo-1-in dan yl)acetic acid (15b):
brown oil (13.7 g, 97%); 1H NMR δ (300 MHz, DMSO-d6) 1.59
(s, 3H, CH3), 2.65 and 3.05 (d, 2H, J ) 16 Hz, CH2), 2.94 and
3.13 (d, 2H, J ) 14 Hz, CH2), 7.62 (d, 1H, J ) 2 Hz, ArH),
7.90 (d, 1H, J ) 2 Hz, ArH), 12.20 (br s, 1H, CO2H).
5,6-Dich lor o-1-m eth yl-3-oxo-1-in dan yl)acetic acid (15c)
a n d (4,5-d ich lor o-1-m et h yl-3-oxo-1-in d a n yl)a cet ic a cid
(15d ) m ixtu r e: brown solid (49 g, 100%), 60/40 isomeric
mixture; 1H NMR δ (300 MHz, DMSO-d6) major isomer
(compound 15c): 1.42 (s, 3H, CH3), 2.55-3.00 (m, 4H, 2CH2),
7.75 (s, 1H, ArH), 8.10 (s, 1H, ArH), 12.10 (br s, 1H, CO2H);
minor isomer (compound 15d ): 1.42 (s, 3H, CH3), 2.55-3.00
(m, 4H, 2CH2), 7.70 (d, 1H, J ) 7 Hz, ArH), 7.88 (d, 1H, J )
7 Hz, ArH), 12.10 (br s, 1H, CO2H).
5-F lu or o-1-m eth yl-3-oxo-1-in d a n yl)a cetic a cid (15e):
beige solid (24.5 g, 100%); 1H NMR δ (250 MHz, DMSO-d6)
1.45 (s, 3H, CH3), 2.59 and 3.00 (d, 2H, J ) 16 Hz, CH2), 2.78
(m, 2H, CH2), 7.37 (dd, 1H, J ) 7 and 2 Hz, ArH), 7.59 (dt,
1H, J ) 7 and 2 Hz, ArH), 7.82 (dd, 1H, J ) 7 and 4 Hz, ArH),
12.10 (br s, 1H, CO2H).
5-Tr iflu or om et h oxy-1-m et h yl-3-oxo-1-in d a n yl)a cet ic
a cid (15f): brown oil (8 g, 53%); 1H NMR δ (250 MHz, DMSO-
d6) 1.45 (s, 3H, CH3), 2.59 and 3.02 (d, 2H, J ) 16 Hz, CH2),
2.82 (m, 2H, CH2), 7.50 (m, 1H, ArH), 7.71 (d, 1H, J ) 7 Hz,
ArH), 7.90 (d, 1H, J ) 7 Hz, ArH).
5-Ch lor o-1-m eth yl-3-oxo-1-in d a n yl)a cetic a cid (15g):
beige solid (51 g, 58%), mp 119 °C; 1H NMR δ (300 MHz,
DMSO-d6) 1.42 (s, 3H, CH3), 2.55 and 2.97 (d, 2H, J ) 16 Hz,
CH2), 2.80 (m, 2H, CH2), 7.56 (d, 1H, J ) 2 Hz, ArH), 7.75 (m,
2H, ArH), 12.10 (br s, 1H, CO2H).
7.48 (t, 1H, J ) 7 Hz, ArH), 7.60 (d, 1H, J ) 7 Hz, ArH), 7.71
(d, 1H, J ) 7 Hz, ArH).
Compounds 16b-i were obtained following procedure E.
Eth yl (5,7-d ich lor o-1-m eth yl-3-oxo-1-in d a n yl)a ceta te
1
(16b): 15 g (91%); H NMR δ (250 MHz, DMSO-d6) 0.97 (t,
3H, J ) 6 Hz, CH3), 1.55 (s, 3H, CH3), 2.65 and 3.00 (d, 2H, J
) 16 Hz, CH2), 2.95 and 3.15 (d, 2H, J ) 14 Hz, CH2), 3.85 (q,
2H, J ) 6 Hz, OCH2), 7.59 (d, 1H, J ) 2 Hz, ArH), 7.88 (d,
1H, J ) 2 Hz, ArH).
Eth yl (5,6-d ich lor o-1-m eth yl-3-oxo-1-in d a n yl)a ceta te
(16c) a n d eth yl (4,5-d ich lor o-1-m eth yl-3-oxo-1-in d a n y-
l)a ceta te (16d ) were prepared starting from the mixture of
15c and 15d and separated by flash chromatography on silica
gel using a mixture of cyclohexane and ethyl acetate (v/v )
80/20) as eluent.
1
Com p ou n d 16c: orange oil, 16.8 g (33%); H NMR δ (300
MHz, DMSO-d6) 1.00 (t, 3H, J ) 6 Hz, CH3), 1.42 (s, 3H, CH3),
2.55 and 2.92 (d, 2H, J ) 16 Hz, CH2), 2.90 (m, 2H, CH2), 3.90
(q, 2H, J ) 6 Hz, OCH2), 7.75 (s, 1H, ArH), 8.15 (s, 1H, ArH).
Com p ou n d 16d : yellow oil, 4.6 g (9%); 1H NMR δ (300
MHz, DMSO-d6) 1.00 (t, 3H, J ) 6 Hz, CH3), 1.41 (s, 3H, CH3),
2.60 and 2.95 (d, 2H, J ) 16 Hz, CH2), 2.85 (m, 2H, CH2), 3.90
(q, 2H, J ) 6 Hz, OCH2), 7.73 (d, 1H, J ) 7 Hz, ArH), 7.90 (d,
1H, J ) 7 Hz, ArH).
Eth yl (1,5-d im eth yl-3-oxo-1-in d a n yl)a ceta te (16e) (us-
ing (1,5-dimethyl-3-oxo-1-indanyl)acetic acid as starting mate-
1
rial):15 20.5 g (92%); H NMR δ (300 MHz, DMSO-d6) 0.98 (t,
3H, J ) 6 Hz, CH3), 1.41 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.50
and 2.95 (d, 2H, J ) 16 Hz, CH2), 2.80 (m, 2H, CH2), 3.90 (q,
2H, J ) 6 Hz, OCH
2), 7.40 (s, 1H, ArH), 7.50 (d, 1H, J ) 7 Hz,
ArH), 7.60 (d, 1H, J ) 7 Hz, ArH).
Eth yl (5-flu or o-1-m eth yl-3-oxo-1-in d a n yl)a ceta te (16f):
20.9 g (91%); 1H NMR δ (300 MHz, DMSO-d6) 1.00 (t, 3H, J )
6 Hz, CH3), 1.45 (s, 3H, CH3), 2.58 and 2.98 (d, 2H, J ) 16 Hz,
CH2), 2.85 (m, 2H, CH2), 3.90 (q, 2H, J ) 6 Hz, OCH2), 7.32
(dd, 1H, J ) 7 and 2 Hz, ArH), 7.55 (dt, 1H, J ) 7 and 2 Hz,
ArH), 7.82 (dd, 1H, J ) 7 and 4 Hz, ArH).
Eth yl (5-tr iflu or om eth oxy-1-m eth yl-3-oxo-1-in d a n yl)-
1
a ceta te (16g): 5.7 g (65%); H NMR δ (300 MHz, DMSO-d6)
0.98 (t, 3H, J ) 6 Hz, CH3), 1.45 (s, 3H, CH3), 2.59 and 3.00
(d, 2H, J ) 16 Hz, CH2), 2.88 (m, 2H, CH2), 3.90 (q, 2H, J )
6 Hz, OCH2), 7.48 (d, 1H, J ) 2 Hz, ArH), 7.70 (dd, 1H, J ) 7
and 2 Hz, ArH), 7.90 (d, 1H, J ) 7 Hz, ArH).
Eth yl (5-ch lor o-1-m eth yl-3-oxo-1-in dan yl)acetate (16h ):
1
43 g (97%); H NMR δ (300 MHz, DMSO-d6) 1.00 (t, 3H, J )
6 Hz, CH3), 1.42 (s, 3H, CH3), 2.56 and 2.97 (d, 2H, J ) 16 Hz,
CH2), 2.85 (m, 2H, CH2), 3.90 (q, 2H, J ) 6 Hz, OCH2), 7.56
(d, 1H, J ) 2 Hz, ArH), 7.75 (m, 2H, ArH).
Eth yl (5-ch lor o-1-p r op yl-3-oxo-1-in d a n yl)a ceta te (16i):
13.2 g (94%); IR (60 g/L CCl4 solution, cm-1) 3000-2850 (ν
CH2 and CH3); 1725 (ν CdO ester and ketone); 1220 (ν C-O
ester); 830 (γ aromatic CH).
5-Ch lor o-1-p r op yl-3-oxo-1-in d a n yl)a cetic a cid (15h ):
1
Gen er al P r ocedu r e F. Eth yl (7-Ch lor o-2-h ydr oxyim in o-
1-m eth yl-3-oxo-1-in d a n yl)a ceta te (17a ). tert-Butyl nitrite
(7.8 mL, 0.066 mol) is added to a solution of 12 g (0.045 mol)
of ethyl (7-chloro-1-methyl-3-oxo-1-indanyl)acetate (16a ) in 50
mL of diethyl ether and 18 mL of a 5 N ethereal hydrogen
chloride solution cooled to 5 °C. The reaction mixture is stirred
for 1 h at room temperature and then concentrated to dryness
under reduced pressure. The oily residue obtained is taken
up in ethyl acetate and evaporated to dryness under reduced
pressure. The latter operation is carried out four times in order
to remove the excess tert-butyl nitrite by azeotropic distillation;
12.4 g (93%) of ethyl (7-chloro-2-hydroxyimino-1-methyl-3-oxo-
1-indanyl)acetate hydrochloride (17a ) is thereby obtained in
white powder (9.6 g, 75%), mp 136 °C; H NMR δ (300 MHz,
DMSO-d6) 0.78 (t, 3H, J ) 7 Hz, CH3), 0.80 and 1.18 (m, 2H,
CH2), 1.66 and 1.80 (m, 2H, CH2), 2.63 and 2.90 (d, 2H, J )
16 Hz, CH2), 2.80 (m, 2H, CH2), 7.59 (d, 1H, J ) 2 Hz, ArH),
7.75 (m, 2H, ArH).
Gen er a l P r oced u r e E. Eth yl (7-Ch lor o-1-m eth yl-3-oxo-
1-in d a n yl)a ceta te (16a ). Oxalyl chloride (4.7 mL, 0.055 mol)
is added slowly to a mixture of 11.6 g (0.05 mol) of (7-chloro-
1-methyl-3-oxo-1-indanyl)acetic acid (15a ) and 0.2 mL of N,N-
dimethylformamide in 150 mL of dichloromethane. After a 4
h stirring at room temperature, 30 mL of ethanol is added
slowly. The reaction mixture is then further stirred for 12 h
and washed twice with 100 mL of a saturated aqueous solution
of sodium hydrogencarbonate and 100 mL of saturated aque-
ous solution of sodium chloride, dried over magnesium sulfate,
and concentrated to dryness under reduced pressure; 12 g
(90%) of ethyl (7-chloro-1-methyl-3-oxo-1-indanyl)acetate (16a )
is thereby obtained in the form of an orange oil, which is used
in subsequent suntheses without further purification. 1H NMR
δ (300 MHz, DMSO-d6) 0.95 (t, 3H, J ) 6 Hz, CH3), 1.58 (s,
3H, CH3), 2.62 and 3.00 (d, 2H, J ) 16 Hz, CH2), 2.98 and
3.16 (d, 2H, J ) 14 Hz, CH2), 3.85 (q, 2H, J ) 6 Hz, OCH2),
1
the form of a yellow solid melting at 115 °C. H NMR δ (300
MHz, DMSO-d6) 0.85 (t, 3H, J ) 6 Hz, CH3), 1.73 (s, 3H, CH3),
2.95 and 3.65 (d, 2H, J ) 14 Hz, CH2), 3.75 (q, 2H, J ) 6 Hz,
OCH2), 7.52 (t, 1H, J ) 7 Hz, ArH), 7.75 (d, 1H, J ) 7 Hz,
ArH), 7.78 (d, 1H, J ) 7 Hz, ArH), 12.85 (s, 1H, NOH).
Compounds 17b-i were obtained following procedure F.
Eth yl (5,7-d ich lor o-2-h yd r oxyim in o-1-m eth yl-3-oxo-1-
in d a n yl)a ceta te h yd r och lor id e (17b): beige crystals (15.5
1
g, 98%), mp 137 °C; H NMR δ (300 MHz, DMSO-d6) 0.92 (t,

Indeno[1,2-b]pyrazin-2,3-diones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2377
3H, J ) 6 Hz, CH3), 1.77 (s, 3H, CH3), 3.47 and 3.69 (d, 2H, J
) 14 Hz, CH2), 3.82 (q, 2H, J ) 6 Hz, OCH2), 7.80 (d, 1H, J )
2 Hz, ArH), 8.02 (d, 1H, J ) 2 Hz, ArH), 13.00 (s, 1H, NOH).
Eth yl (5,6-d ich lor o-2-h yd r oxyim in o-1-m eth yl-3-oxo-1-
in d a n yl)a ceta te h yd r och lor id e (17c): white crystals (14.2
d6) major compound: 1.06 (t, 3H, ethyl CH3), 1.36 (s, 3H, CH3),
3.20 and 3.46 (d, 2H, J ) 16 Hz, CH2), 3.96 (m, 2H, OCH2),
4.50 (s, 1H, CH), 7.40-7.90 (m, 4H, ArH), 8.94 (br s, 3H,
NH3+Cl-); minor compound: 0.86 (t, 3H, ethyl CH3), 1.75 (s,
3H, CH3), 2.92 (m, 2H, CH2), 3.80 (m, 2H, OCH2), 4.22 (s, 1H,
CH), 7.40-7.90 (m, 4H, ArH), 8.94 (br s, 3H, NH3+Cl-).
1
g, 86%), mp 216 °C; H NMR δ (300 MHz, DMSO-d6) 0.95 (t,
3H, J ) 6 Hz, CH3), 1.60 (s, 3H, CH3), 3.28 and 3.58 (d, 2H, J
) 14 Hz, CH2), 3.83 (q, 2H, J ) 6 Hz, OCH2), 8.00 (s, 1H, ArH),
8.30 (s, 1H, ArH), 12.80 (s, 1H, NOH).
Eth yl (2-a m in o-5,7-d ich lor o-1-m eth yl-3-oxo-1-in d a n yl)-
a ceta te h yd r och lor id e (18c): 13.2 g (89%), 60/40 diastere-
oisomeric mixture; 1H NMR δ (300 MHz, DMSO-d6) major
compound: 1.05 (t, 3H, J ) 6 Hz, CH3), 1.47 (s, 3H, CH3), 3.37
and 3.65 (d, 2H, J ) 14 Hz, CH2), 3.96 (q, 2H, J ) 6 Hz, OCH2),
4.52 (s, 1H, CH), 7.50-8.00 (m, 2H, ArH), 9.20 (br s, 3H,
NH3+Cl-); minor compound: 0.85 (t, 3H, J ) 6 Hz, CH3), 1.87
(s, 3H, CH3), 3.20 (m, 2H, CH2), 3.80 (q, 2H, J ) 6 Hz, OCH2),
4.35 (s, 1H, CH), 7.50-8.00 (m, 2H, ArH), 9.20 (br s, 3H,
NH3+Cl-).
Eth yl (2-a m in o-5,6-d ich lor o-1-m eth yl-3-oxo-1-in d a n yl)-
a ceta te h yd r och lor id e (18d ): 8.2 g (100%), 70/30 diastere-
oisomeric mixture; 1H NMR δ (300 MHz, DMSO-d6) major
compound: 1.07 (t, 3H, J ) 6 Hz, CH3), 1.45 (s, 3H, CH3), 3.30
and 3.52 (d, 2H, J ) 14 Hz, CH2), 3.96 (q, 2H, J ) 6 Hz, OCH2),
4.47 (s, 1H, CH), 8.02 (s, 1H, ArH), 8.26 (s, 1H, ArH), 9.15 (br
s, 3H, NH3+Cl-); minor compound: 0.86 (t, 3H, J ) 6 Hz, CH3),
1.75 (s, 3H, CH3), 3.05 (m, 2H, CH2), 3.80 (q, 2H, J ) 6 Hz,
OCH2), 4.27 (s, 1H, CH), 7.97 (s, 1H, ArH), 8.19 (s, 1H, ArH),
9.15 (br s, 3H, NH3+Cl-).
Eth yl (2-a m in o-4,5-d ich lor o-1-m eth yl-3-oxo-1-in d a n yl)-
a ceta te h yd r och lor id e (18e): 4.3 g (100%), 70/30 diastere-
oisomeric mixture; 1H NMR δ (300 MHz, DMSO-d6) major
compound: 1.07 (t, 3H, J ) 6 Hz, CH3), 1.40 (s, 3H, CH3), 3.29
and 3.55 (d, 2H, J ) 14 Hz, CH2), 3.97 (q, 2H, J ) 6 Hz, OCH2),
4.55 (s, 1H, CH), 7.83 (d, 1H, J ) 7 Hz, ArH), 8.07 (d, 1H, J )
7 Hz, ArH), 9.30 (br s, 3H, NH3+Cl-); minor compound: 0.86
(t, 3H, J ) 6 Hz, CH3), 1.75 (s, 3H, CH3), 3.05 (m, 2H, CH2),
3.80 (q, 2H, J ) 6 Hz, OCH2), 4.27 (s, 1H, CH), 7.77 (d, 1H, J
) 7 Hz, ArH), 8.03 (d, 1H, J ) 7 Hz, ArH), 9.30 (br s, 3H,
NH3+Cl-).
E t h yl (2-a m in o-1,5-d im et h yl-3-oxo-1-in d a n yl)a cet a t e
h yd r och lor id e (18f): 21 g (100%), 60/40 diastereoisomeric
mixture; 1H NMR δ (300 MHz, DMSO-d6) major compound:
1.05 (t, 3H, J ) 6 Hz, CH3), 1.38 (s, 3H, CH3), 2.40 (s, 3H,
CH3), 3.18 and 3.52 (d, 2H, J ) 14 Hz, CH2), 3.93 (q, 2H, J )
6 Hz, OCH2), 4.50 (s, 1H, CH), 7.50-7.70 (m, 3H, ArH), 9.10
(br s, 3H, NH3+Cl-); minor compound: 0.90 (t, 3H, J ) 6 Hz,
CH3), 1.75 (s, 3H, CH3), 2.40 (s, 3H, CH3), 2.95 (m, 2H, CH2),
3.80 (q, 2H, J ) 6 Hz, OCH2), 4.17 (s, 1H, CH), 7.50-7.70 (m,
3H, ArH), 9.10 (br s, 3H, NH3+Cl-).
Eth yl (4,5-d ich lor o-2-h yd r oxyim in o-1-m eth yl-3-oxo-1-
in d a n yl)a ceta te h yd r och lor id e (17d ): white crystals (3.5
1
g, 71%), mp 198 °C; H NMR δ (250 MHz, DMSO-d6) 0.96 (t,
3H, J ) 6 Hz, CH3), 1.60 (s, 3H, CH3), 3.25 and 3.58 (d, 2H, J
) 14 Hz, CH2), 3.81 (q, 2H, J ) 6 Hz, OCH2), 7.85 (d, 1H, J )
7 Hz, ArH), 8.05 (d, 1H, J ) 7 Hz, ArH), 12.75 (s, 1H, NOH).
Eth yl (5-m eth yl-2-h yd r oxyim in o-1-m eth yl-3-oxo-1-in -
d a n yl)a ceta te h yd r och lor id e (17e): white crystals (18 g,
81%), mp 187 °C; 1H NMR δ (300 MHz, DMSO-d6) 0.91 (t, 3H,
J ) 6 Hz, CH3), 1.51 (s, 3H, CH3), 2.40 (s, 3H, CH3), 3.08 and
3.51 (d, 2H, J ) 14 Hz, CH2), 3.72 (q, 2H, J ) 6 Hz, OCH2),
7.50 (s, 1H, ArH), 7.55 (d, 1H, J ) 7 Hz, ArH), 7.63 (d, 1H, J
) 7 Hz, ArH), 12.40 (s, 1H, NOH).
Eth yl (5-flu or o-2-h yd r oxyim in o-1-m eth yl-3-oxo-1-in -
d a n yl)a ceta te h yd r och lor id e (17f): beige crystals (25.5 g,
97%), mp 122 °C; 1H NMR δ (300 MHz, DMSO-d6) 0.92 (t, 3H,
J ) 6 Hz, CH3), 1.60 (s, 3H, CH3), 3.18 and 3.57 (d, 2H, J )
14 Hz, CH2), 3.80 (q, 2H, J ) 6 Hz, OCH2), 7.53 (dd, 1H, J )
7 and 2 Hz, ArH), 7.67 (dt, 1H, J ) 7 and 2 Hz, ArH), 7.88
(dd, 1H, J ) 7 and 4 Hz, ArH), 12.70 (s, 1H, NOH).
Eth yl (5-tr iflu or om eth oxy-2-h yd r oxyim in o-1-m eth yl-
3-oxo-1-in d a n yl)a ceta te h yd r och lor id e (17g): beige crys-
tals (3.1 g, 50%), mp 154 °C; 1H NMR δ (300 MHz, DMSO-d6)
0.88 (t, 3H, J ) 6 Hz, CH3), 1.60 (s, 3H, CH3), 3.25 and 3.55
(d, 2H, J ) 14 Hz, CH2), 3.75 (q, 2H, J ) 6 Hz, OCH2), 7.65 (d,
1H, J ) 2 Hz, ArH), 7.80 (dd, 1H, J ) 7 and 2 Hz, ArH), 7.97
(d, 1H, J ) 7 Hz, ArH), 12.73 (s, 1H, NOH).
Eth yl (5-ch lor o-2-h yd r oxyim in o-1-m eth yl-3-oxo-1-in -
d a n yl)a ceta te h yd r och lor id e (17h ): yellow solid (43 g,
1
97%); H NMR δ (300 MHz, DMSO-d6) 0.92 (t, 3H, J ) 6 Hz,
CH3), 1.58 (s, 3H, CH3), 3.20 and 3.56 (d, 2H, J ) 14 Hz, CH2),
3.78 (q, 2H, J ) 6 Hz, OCH2), 7.76 (d, 1H, J ) 2 Hz, ArH),
7.85 (m, 2H, ArH), 12.70 (s, 1H, NOH).
Eth yl (5-ch lor o-2-h yd r oxyim in o-1-p r op yl-3-oxo-1-in -
d a n yl)a ceta te h yd r och lor id e (17i): off-white powder (13.85
g, 95%), mp 144 °C; 1H NMR δ (250 MHz, DMSO-d6) 0.55 and
0.80 (m, 2H, CH2), 0.75 (t, 3H, J ) 6 Hz, CH3), 0.95 (t, 3H, J
) 6 Hz, CH3), 1.85 and 2.40 (m, 2H, CH2), 3.18 and 3.55 (d,
2H, J ) 14 Hz, CH2), 3.78 (q, 2H, J ) 6 Hz, OCH2), 7.78 (s,
1H, ArH), 7.87 (m, 2H, ArH), 12.75 (s, 1H, NOH).
Eth yl (2-a m in o-5-flu or o-1-m eth yl-3-oxo-1-in d a n yl)a c-
eta te h yd r och lor id e (18g): 31 g (100%), 60/40 diastereoi-
someric mixture; 1H NMR δ (300 MHz, DMSO-d6) major
compound: 1.05 (t, 3H, J ) 6 Hz, CH3), 1.40 (s, 3H, CH3), 3.19
and 3.50 (d, 2H, J ) 14 Hz, CH2), 3.93 (q, 2H, J ) 6 Hz, OCH2),
4.52 (s, 1H, CH), 7.40-7.90 (m, 3H, ArH), 9.10 (br s, 3H,
NH3+Cl-); minor compound: 0.90 (t, 3H, J ) 6 Hz, CH3), 1.75
(s, 3H, CH3), 2.98 (m, 2H, CH2), 3.80 (q, 2H, J ) 6 Hz, OCH2),
4.25 (s, 1H, CH), 7.40-7.90 (m, 3H, ArH), 9.00 (br s, 3H,
NH3+Cl-).
Gen er a l P r oced u r e G. E t h yl (2-Am in o-7-ch lor o-1-
m eth yl-3-oxo-1-in d a n yl)a ceta te Hyd r och lor id e (18b). A
solution of 12 g (0.04 mol) of ethyl (7-chloro-2-hydroxyimino-
1-methyl-3-oxo-1-indanyl)acetate hydrochloride (17a ) in 200
mL of acetic acid is saturated with gaseous hydrochloric acid,
and the mixture is then hydrogenated for 20 h at a pressure
of 1.8 bar of hydrogen at room temperature in the presence of
2 g of palladium on charcoal (palladium content 10%). The
reaction mixture is filtered and the filtrate concentrated under
reduced pressure; 11.4 g (97%) of ethyl (2-amino-7-chloro-1-
methyl-3-oxo-1-indanyl)acetate hydrochloride (18b) is obtained
in the form of a light brown solid as a 60/40 diastereoisomeric
mixture; 1H NMR δ (300 MHz, DMSO-d6) major compound:
1.05 (t, 3H, J ) 6 Hz, CH3), 1.94 (s, 3H, CH3), 3.43 and 3.62
(d, 2H, J ) 14 Hz, CH2), 3.98 (q, 2H, J ) 6 Hz, OCH2), 4.50 (s,
1H, CH), 7.55-7.90 (m, 3H, ArH), 9.20 (br s, 3H, NH3+Cl-);
minor compound: 0.87 (t, 3H, J ) 6 Hz, CH3), 1.53 (s, 3H,
CH3), 3.22 (m, 2H, CH2), 3.59 (q, 2H, J ) 6 Hz, OCH2), 4.34
(s, 1H, CH), 7.55-7.90 (m, 3H, ArH), 9.20 (br s, 3H, NH3+Cl-).
Compounds 18a ,c-j were obtained following procedure G
starting from the corresponding material.
Eth yl (2-a m in o-5-tr iflu or om eth oxy-1-m eth yl-3-oxo-1-
in d a n yl)a ceta te h yd r och lor id e (18h ): 3.7 g (100%), 55/45
1
diastereoisomeric mixture; H NMR δ (300 MHz, DMSO-d6)
major compound: 1.03 (t, 3H, J ) 6 Hz, CH3), 1.47 (s, 3H,
CH3), 3.27 and 3.55 (d, 2H, J ) 14 Hz, CH2), 3.95 (q, 2H, J )
6 Hz, OCH2), 4.54 (s, 1H, CH), 7.64 (s, 1H, ArH), 7.80-8.00
(m, 2H, ArH), 9.20 (br s, 3H, NH3+Cl-); minor compound: 0.88
(t, 3H, J ) 6 Hz, CH3), 1.80 (s, 3H, CH3), 2.98 and 3.10 (d, 2H,
J ) 14 Hz, CH2), 3.80 (q, 2H, J ) 6 Hz, OCH2), 4.32 (s, 1H,
CH), 7.69 (s, 1H, ArH), 7.80-8.00 (m, 2H, ArH), 9.20 (br s,
3H, NH3+Cl-).
Eth yl (2-a m in o-5-ch lor o-1-m eth yl-3-oxo-1-in d a n yl)a c-
eta te h yd r och lor id e (18i): 10.5 g (100%), 65/35 diastereoi-
someric mixture; 1H NMR δ (250 MHz, DMSO-d6) major
compound: 1.05 (t, 3H, J ) 6 Hz, CH3), 1.40 (s, 3H, CH3), 3.25
and 3.55 (d, 2H, J ) 14 Hz, CH2), 3.95 (q, 2H, J ) 6 Hz, OCH2),
4.50 (s, 1H, CH), 7.70-7.90 (m, 3H, ArH), 9.30 (br s, 3H,
Eth yl (2-a m in o-1-m eth yl-3-oxo-1-in d a n yl)a ceta te h y-
d r och lor id e (18a ) (prepared from ethyl (2-hydroxyimino-1-
methyl-3-oxo-1-indanyl)acetate hydrochloride):16 11.4 g (97%),
60/40 diastereoisomeric mixture; 1H NMR δ (300 MHz, DMSO-

2378 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
J imonet et al.
NH3+Cl-); minor compound: 0.92 (t, 3H, J ) 6 Hz, CH3), 1.76
(s, 3H, CH3), 2.95 and 3.07 (d, 2H, J ) 14 Hz, CH2), 3.81 (q,
2H, J ) 6 Hz, OCH2), 4.27 (s, 1H, CH), 7.70-7.90 (m, 3H,
ArH), 9.30 (br s, 3H, NH3+Cl-).
Eth yl (2-a m in o-5-ch lor o-1-p r op yl-3-oxo-1-in d a n yl)a c-
eta te h yd r och lor id e (18j): 0.29 g (93%), 65/35 diastereoi-
someric mixture; 1H NMR δ (200 MHz, DMSO-d6) major
compound: 0.70-2.20 (m, 7H, propyl), 2.88 and 3.04 (d, 2H,
J ) 14 Hz, CH2), 3.80 (q, 2H, J ) 6 Hz, OCH2), 4.25 (s, 1H,
CH), 7.70-7.90 (m, 3H, ArH), 8.75 (br s, 3H, NH3+Cl-); minor
compound: 0.70-2.20 (m, 7H, propyl), 3.25 (m, 2H, CH2), 4.01
(q, 2H, J ) 6 Hz, OCH2), 4.63 (s, 1H, CH), 7.70-7.90 (m, 3H,
ArH), 8.75 (br s, 3H, NH3+Cl-).
(d, 1H, J ) 9 Hz, NH); minor compound: 0.92 (t, 3H, J ) 6
Hz, CH3), 1.30 (t, 3H, J ) 6 Hz, CH3), 1.60 (s, 3H, CH3), 2.80
(m, 2H, CH2), 3.83 (m, 2H, OCH2), 4.30 (q, 2H, J ) 6 Hz,
OCH2), 4.89 (d, 1H, J ) 9 Hz, CH), 7.96 (s, 1H, ArH), 8.17 (s,
1H, ArH), 8.30 (d, 1H, J ) 9 Hz, NH).
Eth yl N-(4,5-dich lor o-1-eth oxyca r bon ylm eth yl-1-m eth -
yl-3-oxo-2-in d a n yl)oxa m a te (19e): 2.6 g (52%), 55/45 dias-
tereoisomeric mixture; 1H NMR δ (300 MHz, DMSO-d6) major
compound: 1.05 (t, 3H, J ) 6 Hz, CH3), 1.20 (s, 3H, CH3), 1.30
(t, 3H, J ) 6 Hz, CH3), 2.90 and 3.18 (d, 2H, J ) 14 Hz, CH2),
3.95 (m, 2H, OCH2), 4.30 (q, 2H, J ) 6 Hz, OCH2), 5.05 (d,
1H, J ) 9 Hz, CH), 7.76 (d, 1H, J ) 7 Hz, ArH), 8.08 (d, 1H,
J ) 7 Hz, ArH), 9.35 (d, 1H, J ) 9 Hz, NH); minor compound:
0.90 (t, 3H, J ) 6 Hz, CH3), 1.30 (t, 3H, J ) 6 Hz, CH3), 1.59
(s, 3H, CH3), 2.77 (m, 2H, CH2), 3.85 (m, 2H, OCH2), 4.30 (q,
2H, J ) 6 Hz, OCH2), 4.92 (d, 1H, J ) 9 Hz, CH), 7.76 (d, 1H,
J ) 7 Hz, ArH), 8.10 (d, 1H, J ) 7 Hz, ArH), 8.40 (d, 1H, J )
9 Hz, NH).
Gen er a l P r oced u r e H. Eth yl N-(7-Ch lor o-1-eth oxyca r -
bon ylm eth yl-1-m eth yl-3-oxo-2-in d a n yl)oxa m a te (19b). A
mixture of 11 g (0.04 mol) of ethyl (2-amino-7-chloro-1-methyl-
3-oxo-1-indanyl)acetate hydrochloride (18b) and 150 mL of
dichloromethane is cooled to 0 °C. Ethyl oxalyl chloride (5 mL,
0.045 mol) is then added, followed by a slow addition of 11
mL (0.08 mol) of triethylamine while the temperature of the
reaction medium is maintained close to 0 °C. When the
addition is complete, the temperature of the reaction mixture
is allowed to rise to about 20 °C. The mixture is then filtered
and the filtrate is washed with distilled water (2 × 80 mL).
The organic solution is dried over magnesium sulfate, filtered,
and evaporated in a rotary evaporator to give 14 g of crude
product purified by flash chromatography on silica gel using
a mixture of cyclohexane and ethyl acetate (60/40) as eluent;
10 g (67%) of ethyl N-(7-chloro-1-ethoxycarbonylmethyl-1-
methyl-3-oxo-2-indanyl)oxamate (19b) is thus obtained in the
Eth yl N-(5-m eth yl-1-eth oxyca r bon ylm eth yl-1-m eth yl-
3-oxo-2-in d a n yl)oxa m a te (19f): 16.8 g (76%), 55/45 diaste-
1
reoisomeric mixture; H NMR δ (300 MHz, DMSO-d6) major
compound: 0.88 (t, 3H, J ) 6 Hz, CH3), 1.30 (t, 3H, J ) 6 Hz,
CH3), 1.59 (s, 3H, CH3), 2.40 (s, 3H, CH3), 2.65 and 2.75 (d,
2H, J ) 14 Hz, CH2), 3.79 (m, 2H, OCH2), 4.30 (m, 2H, OCH2),
4.76 (d, 1H, J ) 9 Hz, CH), 7.50-7.70 (m, 3H, ArH), 8.50 (d,
1H, J ) 9 Hz, NH); minor compound: 1.03 (t, 3H, J ) 6 Hz,
CH3), 1.18 (s, 3H, CH3), 1.30 (t, 3H, J ) 6 Hz, CH3), 2.40 (s,
3H, CH3), 2.90 and 3.06 (d, 2H, J ) 14 Hz, CH2), 3.93 (m, 2H,
OCH2), 4.30 (m, 2H, OCH2), 5.08 (d, 1H, J ) 9 Hz, CH), 7.50-
7.70 (m, 3H, ArH), 9.30 (d, 1H, J ) 9 Hz, NH).
1
form of a yellow oil as a 55/45 diastereoisomeric mixture. H
Eth yl N-(5-flu or o-1-eth oxyca r bon ylm eth yl-1-m eth yl-
3-oxo-2-in d a n yl)oxa m a te (19g): 24 g (83%), 60/40 diaste-
NMR δ (300 MHz, DMSO-d6) major compound: 0.81 (t, 3H, J
) 6 Hz, CH3), 1.32 (s, 3H, CH3), 1.30 (t, 3H, J ) 6 Hz, CH3),
3.00 and 3.38 (d, 2H, J ) 14 Hz, CH2), 3.72 (m, 2H, OCH2),
4.30 (q, 2H, J ) 6 Hz, OCH2), 4.96 (d, 1H, J ) 9 Hz, CH),
7.50-7.80 (m, 3H, ArH), 8.27 (d, 1H, J ) 9 Hz, NH); minor
compound: 1.02 (t, 3H, J ) 6 Hz, CH3), 1.77 (s, 3H, CH3), 1.30
(t, 3H, J ) 6 Hz, CH3), 2.90 and 3.02 (d, 2H, J ) 14 Hz, CH2),
3.92 (m, 2H, OCH2), 4.30 (q, 2H, J ) 6 Hz, OCH2), 5.08 (d,
1H, J ) 9 Hz, CH), 7.50-7.80 (m, 3H, ArH), 9.42 (d, 1H, J )
9 Hz, NH).
1
reoisomeric mixture; H NMR δ (300 MHz, DMSO-d6) major
compound: 0.90 (t, 3H, J ) 6 Hz, CH3), 1.30 (t, 3H, J ) 6 Hz,
CH3), 1.61 (s, 3H, CH3), 2.68 and 2.79 (d, 2H, J ) 14 Hz, CH2),
3.77 (m, 2H, OCH2), 4.30 (m, 2H, OCH2), 4.85 (d, 1H, J ) 9
Hz, CH), 7.40-7.90 (m, 3H, ArH), 8.45 (d, 1H, J ) 9 Hz, NH);
minor compound: 1.20 (t, 3H, J ) 6 Hz, CH3), 1.21 (s, 3H,
CH3), 1.30 (t, 3H, J ) 6 Hz, CH3), 2.90 and 3.10 (d, 2H, J ) 14
Hz, CH2), 3.90 (m, 2H, OCH2), 4.30 (m, 2H, OCH2), 5.08 (d,
1H, J ) 9 Hz, CH), 7.40-7.90 (m, 3H, ArH), 9.32 (d, 1H, J )
9 Hz, NH).
Compounds 19a ,c-j were obtained following the same
procedure starting from the corresponding material.
Eth yl N-(5-tr iflu or om eth oxy-1-eth oxyca r bon ylm eth yl-
1-m eth yl-3-oxo-2-in d a n yl)oxa m a te (19h ): 3 g (81%), 53/
Eth yl N-(1-eth oxyca r bon ylm eth yl-1-m eth yl-3-oxo-2-in -
d a n yl)oxa m a te (19a ): 0.95 g (91%), 55/45 diastereoisomeric
mixture; 1H NMR δ (200 MHz, DMSO-d6) major compound:
0.88 and 1.34 (2t, 3H each, J ) 7 Hz, ethyl CH3), 1.21 (s, 2H,
CH3), 2.75 (m, 2H, J ) 15 Hz, CH2), 3.92 (m, 2H, OCH2), 4.32
(m, 2H, OCH2), 4.81 (d, 1H, J ) 9 Hz, CH), 7.40-7.85 (m, 4H,
ArH), 8.47 (br d, 1H, J ) 9 Hz, NHCO); minor compound: 1.03
and 1.34 (2t, 3H each, J ) 7 Hz, ethyl CH3), 1.65 (s, 2H, CH3),
3.03 (m, 2H, J ) 16 Hz, CH2), 3.78 (m, 2H, OCH2), 4.32 (m,
2H, OCH2), 5.10 (d, 1H, J ) 9 Hz, CH), 7.40-7.85 (m, 4H,
ArH), 9.29 (br d, 1H, J ) 9 Hz, NHCO).
1
47 diastereoisomeric mixture; H NMR δ (250 MHz, DMSO-
d6) major compound: 0.83 (t, 3H, J ) 6 Hz, CH3), 1.30 (t, 3H,
J ) 6 Hz, CH3), 1.62 (s, 3H, CH3), 2.72 and 2.85 (d, 2H, J )
14 Hz, CH2), 3.78 (m, 2H, OCH2), 4.30 (m, 2H, OCH2), 4.91 (d,
1H, J ) 9 Hz, CH), 7.60-8.00 (m, 3H, ArH), 8.50 (d, 1H, J )
9 Hz, NH); minor compound: 1.03 (t, 3H, J ) 6 Hz, CH3), 1.21
(s, 3H, CH3), 1.30 (t, 3H, J ) 6 Hz, CH3), 2.92 and 3.20 (d, 2H,
J ) 14 Hz, CH2), 3.91 (m, 2H, OCH2), 4.30 (m, 2H, OCH2),
5.08 (d, 1H, J ) 9 Hz, CH), 7.60-8.00 (m, 3H, ArH), 9.35 (d,
1H, J ) 9 Hz, NH).
Eth yl N-(5-ch lor o-1-eth oxyca r bon ylm eth yl-1-m eth yl-
3-oxo-2-in d a n yl)oxa m a te (19i): 5 g (73%), 55/45 diastere-
oisomeric mixture; 1H NMR δ (250 MHz, DMSO-d6) major
compound: 1.03 (t, 3H, J ) 6 Hz, CH3), 1.20 (s, 3H, CH3), 1.30
(t, 3H, J ) 6 Hz, CH3), 2.92 and 3.15 (d, 2H, J ) 14 Hz, CH2),
3.91 (m, 2H, OCH2), 4.30 (m, 2H, OCH2), 5.08 (d, 1H, J ) 9
Hz, CH), 7.65-7.85 (m, 3H, ArH), 9.35 (d, 1H, J ) 9 Hz, NH);
minor compound: 0.88 (t, 3H, J ) 6 Hz, CH3), 1.30 (t, 3H, J
) 6 Hz, CH3), 1.62 (s, 3H, CH3), 2.70 and 2.80 (d, 2H, J ) 14
Hz, CH2), 3.78 (m, 2H, OCH2), 4.30 (m, 2H, OCH2), 4.85 (d,
1H, J ) 9 Hz, CH), 7.65-7.85 (m, 3H, ArH), 8.45 (d, 1H, J )
9 Hz, NH).
E t h yl N-(5-ch lor o-1-et h oxyca r b on ylm et h yl-1-p r op yl-
3-oxo-2-in d a n yl)oxa m a te (19j): 0.61 g (74%); IR (60 g/L
CH2Cl2 solution, cm-1) 3390 (ν NH); 3000-2850 (ν CH2 and
CH3); 1760 and 1710 (ν CdO oxamide); 1730 (ν CdO ester and
ketone); 1515 (δ NH); 830 (γ aromatic CH).
Eth yl N-(5,7-dich lor o-1-eth oxyca r bon ylm eth yl-1-m eth -
yl-3-oxo-2-in d a n yl)oxa m a te (19c): 6.6 g (44%), 55/45 dias-
tereoisomeric mixture; 1H NMR δ (300 MHz, DMSO-d6) major
compound: 0.88 (t, 3H, J ) 6 Hz, CH3), 1.28 (s, 3H, CH3), 1.30
(t, 3H, J ) 6 Hz, CH3), 3.02 and 3.38 (d, 2H, J ) 14 Hz, CH2),
3.78 (m, 2H, OCH2), 4.31 (q, 2H, J ) 6 Hz, OCH2), 5.01 (d,
1H, J ) 9 Hz, CH), 7.75 (d, 1H, J ) 2 Hz, ArH), 8.00 (d, 1H,
J ) 2 Hz, ArH), 8.28 (d, 1H, J ) 9 Hz, NH); minor compound:
1.05 (t, 3H, J ) 6 Hz, CH3), 1.30 (t, 3H, J ) 6 Hz, CH3), 1.75
(s, 3H, CH3), 2.90 and 3.02 (d, 2H, J ) 14 Hz, CH2), 3.97 (m,
2H, OCH2), 4.31 (q, 2H, J ) 6 Hz, OCH2), 5.07 (d, 1H, J ) 9
Hz, CH), 7.78 (d, 1H, J ) 2 Hz, ArH), 7.96 (d, 1H, J ) 2 Hz,
ArH), 9.46 (d, 1H, J ) 9 Hz, NH).
Eth yl N-(5,6-dich lor o-1-eth oxyca r bon ylm eth yl-1-m eth -
yl-3-oxo-2-in d a n yl)oxa m a te (19d ): 6 g (63%), 55/45 dias-
tereoisomeric mixture; 1H NMR δ (300 MHz, DMSO-d6) major
compound: 1.06 (t, 3H, J ) 6 Hz, CH3), 1.20 (s, 3H, CH3), 1.30
(t, 3H, J ) 6 Hz, CH3), 2.92 and 3.23 (d, 2H, J ) 14 Hz, CH2),
3.95 (m, 2H, OCH2), 4.30 (q, 2H, J ) 6 Hz, OCH2), 5.02 (d,
1H, J ) 9 Hz, CH), 7.95 (s, 1H, ArH), 8.16 (s, 1H, ArH), 9.35
Gen er a l P r oced u r e I. Eth yl (6-Ch lor o-5-m eth yl-2,3-
d ioxo-1,4-d ih yd r o-5H -in d e n o[1,2-b]p yr a zin -5-yl)a ce -
ta te (20b). A mixture of 9 g (0.024 mol) of ethyl N-(7-chloro-

Indeno[1,2-b]pyrazin-2,3-diones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2379
1-ethoxycarbonylmethyl-1-methyl-3-oxo-2-indanyl)oxamate (19b)
and 19 g (0.024 mol) of ammonium acetate in 150 mL of acetic
acid is heated to reflux for 4 h. The reaction mixture is then
concentrated under reduced pressure, 100 mL of water is
added, and the mixture is subjected to two extractions with
ethyl acetate (2 × 100 mL). The organic extract is washed with
an aqueous solution of sodium hydrogen carbonate and then
with an aqueous solution of sodium chloride and dried over
The two enantiomers (+)-20i and (-)-20i were prepared in
optically pure form from the racemic compound 20i by
preparative HPLC using a column packed with a chiral
stationary phase (Chiracel OD) and eluting with ethanol; 8.3
g of 20i was submitted to the following conditions: flow-rate,
70 mL/min; detection, UV (254 nm); column diameter, 60 mm;
column length, 32 cm. Enantiomeric excess for both enanti-
omers was evaluated by analytical HPLC using the same
chiral phase (R ) 3.7; Rs ) 3.3).
magnesium sulfate, filtered, and evaporated in
a rotary
(+)-20i: yellow powder (3.8 g, 46%), mp 259 °C; [R]20
)
evaporator. The crude product thus obtained is purified by
crystallization in 50 mL of diethyl ether. After filtration and
drying, 0.7 g (9%) of ethyl (6-chloro-5-methyl-2,3-dioxo-1,4-
dihydro-5H-indeno[1,2-b]pyrazin-5-yl)acetate 20b is obtained
in the form of gray crystals melting at 200 °C. 1H NMR δ (300
MHz, DMSO-d6) 0.70 (t, 3H, J ) 6 Hz, CH3), 1.52 (s, 3H, CH3),
3.09 and 3.37 (d, 2H, J ) 14 Hz, CH2), 3.67 (q, 2H, J ) 6 Hz,
OCH2), 7.10 (d, 1H, J ) 7 Hz, ArH), 7.39 (t, 1H, J ) 7 Hz,
ArH), 7.48 (d, 1H, J ) 7 Hz, ArH).
D
+42.7 ( 1.9 (c ) 0.5, CH3OH); enantiomeric excess >99.5%;
1H NMR δ (300 MHz, DMSO-d6) same description as reported
for racemic 20i.
(-)-20i: yellow powder (4.0 g, 48%), mp 261 °C; [R]20
)
D
-41.6 ( 1.3 (c ) 0.5, CH3OH); enantiomeric excess >99.5%;
1H NMR δ (300 MHz, DMSO-d6) same description as reported
for 20i and (+)-20i.
Eth yl (8-ch lor o-5-p r op yl-2,3-d ioxo-1,4-d ih yd r o-5H-in -
d en o[1,2-b]p yr a zin -5-yl)a ceta te (20j): yellow powder (10.8
Compounds 20a ,c-j were obtained following the same
procedure starting from the corresponding material.
1
g, 73%), mp >260 °C; H NMR δ (300 MHz, DMSO-d6) 0.50
and 0.75 (m, 2H, CH2), 0.68 (t, 3H, J ) 6 Hz, CH3), 0.79 (t,
3H, J ) 6 Hz, CH3), 1.38 (s, 3H, CH3), 1.95 (t, 2H, J ) 6 Hz,
CH2), 3.00 (m, 2H, CH2), 3.72 (q, 2H, J ) 6 Hz, OCH2), 7.18
(dd, 1H, J ) 7 and 2 Hz, ArH), 7.43 (d, 1H, J ) 7 Hz, ArH),
7.53 (d, 1H, J ) 2 Hz, ArH), 12.20 (s, 1H, CONH), 12.25 (s,
1H, CONH).
Eth yl (5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in d en o[1,2-
b]p yr a zin -5-yl)a ceta te (20a ): yellow crystals (0.7 g, 39%),
mp 216 °C; H NMR δ (300 MHz, DMSO-d6) 0.77 (t, 3H, J )
7 Hz, ethyl CH3), 1.40 (s, 3H, CH3), 3.00 (limiting AB, 2H,
CH2), 3.20 (q, 2H, J ) 7 Hz, OCH2), 7.12 and 7.25 (2 t, 2H, J
) 8 Hz, H-6 and H-7), 7.43 and 7.50 (2 d, 2H, J ) 8 Hz, H-5
and H-8).
1
Gen er a l P r oced u r e J . 6-Ch lor o-5-m eth yl-2,3-d ioxo-1,4-
d ih yd r o-5H-in d en o[1,2-b]p yr a zin -5-yl-a cetic Acid (10b).
A mixture of 0.7 g (0.002 mol) of ethyl (6-chloro-5-methyl-2,3-
dioxo-1,4-dihydro-5H-indeno[1,2-b]pyrazin-5-yl)acetate 20b, 30
mL of dioxane, and 10 mL of 8 N hydrochloric acid is heated
to 90 °C for 4 h. The reaction mixture is then evaporated under
reduced pressure, and the evaporation residue is triturated
with a 10 mL mixture of water and ethanol (4/1), filtered off,
and rinsed with distilled water. After drying at 60 °C under
vacuum (1 mmHg; 0.13 kPa), 0.35 g (58%) of (6-chloro-5-
methyl-2,3-dioxo-1,4-dihydro-5H-indeno[1,2-b]pyrazin-5-yl)ace-
tic acid (10b) are obtained in the form of a pale yellow solid
Eth yl (6,8-d ich lor o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-
in d en o[1,2-b]p yr a zin -5-yl)a ceta te (20c): yellow crystals
1
(2.4 g, 50%), mp >260 °C; H NMR δ (200 MHz, DMSO-d6):
0.78 (t, 3H, J ) 6 Hz, CH3), 1.53 (s, 3H, CH3), 3.10 and 3.38
(d, 2H, J ) 14 Hz, CH2), 3.71 (q, 2H, J ) 6 Hz, OCH2), 7.28 (d,
1H, J ) 2 Hz, ArH), 7.52 (d, 1H, J ) 2 Hz, ArH), 12.25 (br s,
2H, 2 CONH).
Eth yl (7,8-d ich lor o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-
in d en o[1,2-b]p yr a zin -5-yl)a ceta te (20d ): yellow crystals
(3.8 g, 79%), mp >260 °C; 1H NMR δ (300 MHz, DMSO-d6)
0.80 (t, 3H, J ) 6 Hz, CH3), 1.43 (s, 3H, CH3), 3.10 (m, 2H,
CH2), 3.75 (q, 2H, J ) 6 Hz, OCH2), 7.75 (s, 1H, ArH), 7.85 (s,
1H, ArH), 12.30 (br s, 2H, 2CONH).
Eth yl (8,9-d ich lor o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-
in d en o[1,2-b]p yr a zin -5-yl)a ceta te (20e): beige crystals
(1.45 g, 63%), mp 249 °C; 1H NMR δ (250 MHz, DMSO-d6)
0.80 (t, 3H, J ) 6 Hz, CH3), 1.49 (s, 3H, CH3), 3.10 (m, 2H,
CH2), 3.75 (q, 2H, J ) 6 Hz, OCH2), 7.42 (d, 1H, J ) 7 Hz,
ArH), 7.50 (d, 1H, J ) 7 Hz, ArH), 10.90 (br s, 1H, CONH),
12.40 (br s, 1H, CONH).
1
melting above 260 °C. H NMR δ (300 MHz, DMSO-d6) 1.50
(s, 3H, CH3), 3.08 and 3.35 (d, 2H, J ) 14 Hz, CH2), 7.13 (d,
1H, J ) 7 Hz, ArH), 7.30 (t, 1H, J ) 7 Hz, ArH), 7.50 (d, 1H,
J ) 7 Hz, ArH), 12.22 (s, 1H, CONH), 12.30 (s, 1H, CONH).
Anal. (C14H11ClN2O4) C, H; N calcd 9.13, found 8.4.
Compounds 10a ,c-j were obtained following the same
procedure starting from the corresponding material.
(5-Me t h yl-2,3-d ioxo-1,4-d ih yd r o-5H -in d e n o[1,2-b]-
p yr a zin -5-yl)a cetic a cid (10a ): pale yellow crystals (0.4 g,
1
59%), mp >260 °C; H NMR δ (300 MHz, DMSO-d6) 1.38 (s,
Eth yl (8-m eth yl-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in -
d en o[1,2-b]p yr a zin -5-yl)a ceta te (20f): yellow crystals (9.6
3H, CH3), 2.90 (s, 2H, CH2), 7.12 (t, 1H, J ) 7 Hz, ArH), 7.25
(t, 1H, J ) 7 Hz, ArH), 7.44 (d, 1H, J ) 7 Hz, ArH), 7.49 (d,
1H, J ) 2 Hz, ArH), 12.20 (s, 1H, CONH), 12.25 (s, 1H,
CONH). Anal. (C14H12N2O4) C, H, N.
1
g, 69%), mp 254 °C; H NMR δ (300 MHz, DMSO-d6) 0.80 (t,
3H, J ) 6 Hz, CH3), 1.40 (s, 3H, CH3), 2.31 (s, 3H, CH3), 2.97
(s, 2H, CH2), 3.72 (q, 2H, J ) 6 Hz, OCH2), 6.96 (d, 1H, J ) 7
Hz, ArH), 7.32 (m, 2H, ArH), 12.15 (br s, 2H, 2CONH).
(6,8-Dich lor o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in d e-
n o[1,2-b]p yr a zin -5-yl)a cetic a cid (10c): yellow crystals
1
Eth yl (8-flu or o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in -
d en o[1,2-b]p yr a zin -5-yl)a ceta te (20g): yellow crystals (3.1
g, 42%), mp >260 °C; 1H NMR δ (200 MHz, DMSO-d6) 0.80 (t,
3H, J ) 6 Hz, CH3), 1.40 (s, 3H, CH3), 3.00 (m, 2H, CH2), 3.75
(q, 2H, J ) 6 Hz, OCH2), 6.94 (m, 1H, ArH), 7.32 (dd, 1H, J )
8 and 2 Hz, ArH), 7.48 (dd, 1H, J ) 8 and 4 Hz, ArH), 12.20
(br s, 2H, 2CONH).
(0.77 g, 84%), mp >260 °C; H NMR δ (300 MHz, DMSO-d6)
1.45 (s, 3H, CH3), 3.05 and 3.30 (d, 2H, J ) 14 Hz, CH2), 7.22
(d, 1H, J ) 2 Hz, ArH), 7.55 (d, 1H, J ) 2 Hz, ArH), 12.30 (br
s, 2H, CONH). Anal. (C14H10Cl2N2O4) C, H, N.
(7,8-Dich lor o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in d e-
n o[1,2-b]p yr a zin -5-yl)a cetic a cid (10d ): yellow crystals (2.5
1
g, 100%), mp >260 °C; H NMR δ (300 MHz, DMSO-d6) 1.40
E t h yl (8-t r iflu or om et h oxy-5-m et h yl-2,3-d ioxo-1,4-d i-
h yd r o-5H-in d en o[1,2-b]p yr a zin -5-yl)a ceta te (20h ): yellow
crystals (0.85 g, 33%), mp 277 °C; 1H NMR δ (300 MHz,
DMSO-d6) 0.78 (t, 3H, J ) 6 Hz, CH3), 1.41 (s, 3H, CH3), 3.05
(s, 2H, CH2), 3.75 (q, 2H, J ) 6 Hz, OCH2), 7.11 (d, 1H, J ) 7
Hz, ArH), 7.57 (m, 2H, ArH), 12.20 (br s, 2H, 2CONH).
(s, 3H, CH3), 2.97 and 3.06 (d, 2H, J ) 14 Hz, CH2), 7.75 (s,
1H, ArH), 7.83 (s, 1H, ArH), 12.25 (br s, 2H, CONH). Anal.
(C14H10Cl2N2O4‚H2O) C, H, N.
(8,9-Dich lor o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in d e-
n o[1,2-b]p yr a zin -5-yl)a cetic a cid (10e): yellow crystals
1
(0.85 g, 94%), mp >260 °C; H NMR δ (250 MHz, DMSO-d6)
1.40 (s, 3H, CH3), 3.03 (m, 2H, CH2), 7.43 (d, 1H, J ) 7 Hz,
ArH), 7.52 (d, 1H, J ) 7 Hz, ArH), 10.90 (br s, 1H, CONH),
12.00 (br s, 1H, CO2H), 12.40 (br s, 1H, CONH). Anal.
(C14H10Cl2N2O4‚H2O) C, H, N.
(5,8-Dim eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in d en o[1,2-b]-
p yr a zin -5-yl)a cetic a cid (10f): yellow crystals (1.27 g, 89%),
mp >260 °C; 1H NMR δ (300 MHz, DMSO-d6) 1.36 (s, 3H,
Eth yl (8-ch lor o-5-m eth yl-2,3-d ioxo-1,4-d ih yd r o-5H-in -
d en o[1,2-b]p yr a zin -5-yl)a ceta te (20i): yellow crystals (5.1
g, 61%), mp 259 °C; H NMR δ (300 MHz, DMSO-d6) 0.78 (t,
3H, J ) 6 Hz, CH3), 1.38 (s, 3H, CH3), 3.00 (m, 2H, CH2), 3.70
(q, 2H, J ) 6 Hz, OCH2), 7.18 (dd, 1H, J ) 7 and 2 Hz, ArH),
7.48 (d, 1H, J ) 7 Hz, ArH), 7.54 (d, 1H, J ) 2 Hz, ArH), 12.20
(s, 1H, CONH), 12.25 (s, 1H, CONH).
1

2380 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
J imonet et al.
CH3), 2.30 (s, 3H, CH3), 2.88 (m, 2H, CH2), 6.95 (d, 1H, J ) 7
Hz, ArH), 7.30 (m, 2H, ArH), 12.00 (br s, 1H, CONH), 12.20
(br s, 1H, CONH). Anal. (C15H14N2O4) C, H, N.
by filtration through Whatman GF/B glass fiber filters, and
filters were immediately rinsed three times with 4 mL of cold
HEPES-KOH buffer (pH 7.5, containing 10 mM magnesium
sulfate). Each determination was performed in duplicate. The
radioactivity remaining on the filters was measured by liquid
scintillometry in Ready-Gel scintillant.
(8-Flu or o-5-m eth yl-2,3-dioxo-1,4-dih ydr o-5H-in den o[1,2-
b]p yr a zin -5-yl)a cet ic a cid (10g): yellow crystals (0.66 g,
1
76%), mp >260 °C; H NMR δ (300 MHz, DMSO-d6) 1.38 (s,
[3H]AMP A Bin d in g. Radiolabeled AMPA binding assays
in rat cerebral cortex membranes were performed as previously
described10b with the following modifications. Briefly, rats were
decapitated and their cerebral cortices were removed on ice
and immediately frozen at -80 C for at least 1 h. The tissue
was rapidly thawed, homogenized with a Polytron in 20
volumes of cold (4 °C) sucrose (0.32 M) and centrifuged at
1000g for 20 min. The supernatant was recentrifuged at
17500g for 20 min at 4 °C. The resulting pellet was suspended
in 50 volumes of ice-cold distilled water, incubated at 37 °C
for 30 min, and centrifuged at 32000g for 20 min at 4 °C. This
procedure was repeated in order to remove any endogenous
glutamate. The biological material thus obtained was sus-
pended in 50 volumes of HEPES buffer (10 mM pH 7.5) and
centrifuged at 32000g for 20 min at 4 °C. The pellet was finally
resuspended in 30 volumes of ice-cold HEPES buffer and was
frozen at -80 °C until use. On the day of the binding assays,
the membranes were thawed and centrifuged at 32000g for
20 min at 4 °C. This procedure was repeated and the final
pellet was suspended in a pH 7.5 buffer containing 10 mM
KH2PO4 and 100 mM KSCN at a concentration of 0.2 mg of
protein/mL. Membranes were then incubated for 30 min at 4
°C with [3H]AMPA (10 nM), the compound under study, or 1
mM L-glutamate for determination of the nonspecific binding.
The binding interaction was terminated by filtration through
glass fiber filters (Printed filtermat A) for Betaplate TM
scintillation counter experiment using a Skatron micro cell
harvester. The filters were immediately rinsed with 5 mL of
cold buffer. The radioactivity remaining on the filters was
measured by liquid scintillometry. Protein levels were mea-
sured by the method of Bradford (Bio-Rad Protein Assay). Each
determination was performed in duplicate.
3H, CH3), 2.93 (m, 2H, CH2), 6.90 (m, 1H, ArH), 7.30 (dd, 1H,
J ) 8 and 2 Hz, ArH), 7.55 (dd, 1H, J ) 8 and 4 Hz, ArH),
12.15 (br s, 1H, CONH), 12.25 (br s, 1H, CONH). Anal.
(C14H11FN2O4) C, H, N.
(5-Met h yl-8-t r iflu or om et h oxy-2,3-d ioxo-1,4-d ih yd r o-
5H-in den o[1,2-b]pyr azin -5-yl)acetic acid (10h ): beige crys-
tals (0.32 g, 66%), mp >260 °C; 1H NMR δ (300 MHz, DMSO-
d6) 1.45 (s, 3H, CH3), 2.98 (m, 2H, CH2), 7.10 (d, 1H, J ) 7 Hz,
ArH), 7.56 (m, 2H, ArH), 11.90 (br s, 1H, CO2H), 12.20 (s, 1H,
CONH), 12.30 (s, 1H, CONH). Anal. (C15H11F3N2O5) C, H; N
calcd 7.86, found 7.2.
(8-Ch lor o-5-m eth yl-2,3-dioxo-1,4-dih ydr o-5H-in den o[1,2-
b]p yr a zin -5-yl)a cetic a cid (10i): yellow powder (1.35 g,
1
75%), mp >260 °C; H NMR δ (300 MHz, DMSO-d6) 1.35 (s,
3H, CH3), 2.93 (m, 2H, CH2), 7.15 (dd, 1H, J ) 7 and 2 Hz,
ArH), 7.45 (d, 1H, J ) 7 Hz, ArH), 7.55 (d, 1H, J ) 2 Hz, ArH),
11.90 (br s, 1H, CO2H), 12.15 (s, 1H, CONH), 12.25 (s, 1H,
CONH). Anal. (C14H11ClN2O4) C, H, N.
(8-Ch lor o-5-m eth yl-2,3-dioxo-1,4-dih ydr o-5H-in den o[1,2-
b]p yr a zin -5-yl)a cetic a cid ((+)-10i) was prepared by start-
ing from (+)-20i: yellow powder (1.5 g, 71%), mp >300 °C;
[R]20D ) +73.2 ( 1.1 (c ) 1, DMF); enantiomeric excess >99.5%
(analytical HPLC using Chiracel OD and 5/95 ethanol/heptane
containing 0.05% trifluoroacetic acid as eluent; R ) 1.17, Rs )
1.43);1H NMR δ (300 MHz, DMSO-d6) same description as
reported for racemic 10i. Anal. (C14H11ClN2O4) C, H, N.
(8-Ch lor o-5-m eth yl-2,3-dioxo-1,4-dih ydr o-5H-in den o[1,2-
b]p yr a zin -5-yl)a cetic a cid ((-)-10i) was prepared by start-
ing from (-)-20i: yellow powder (1.5 g, 71%), mp >300 °C;
[R]20D ) -72.5 ( 1.1 (c ) 1, DMF); enantiomeric excess >99.5%
(see (+)-10i above); 1H NMR δ (300 MHz, DMSO-d6) same
description as reported for 10i and (+)-10i. Anal. (C14H11ClN2O4)
C, H, N.
(8-Ch lor o-5-pr opyl-2,3-dioxo-1,4-dih ydr o-5H-in den o[1,2-
b]p yr a zin -5-yl)a cetic a cid (10j): pale green powder (0.13
g, 40%), mp 262 °C; 1H NMR δ (250 MHz, DMSO-d6) 0.50 and
0.80 (m, 2H, CH2), 1.70 (t, 3H, J ) 6 Hz, CH3), 1.95 (t, 2H, J
) 6 Hz, CH2), 2.95 (s, 2H, CH2), 7.20 (dd, 1H, J ) 7 and 2 Hz,
ArH), 7.45 (d, 1H, J ) 7 Hz, ArH), 7.58 (d, 1H, J ) 2 Hz, ArH),
11.90 (br s, 1H, CO2H), 12.20 (s, 1H, CONH), 12.30 (s, 1H,
CONH). Anal. (C16H15ClN2O4) C, H, N.
Ma xim a l Electr osh ock Assa y. Anticonvulsant activity
was evaluated in mice (CD1 Charles River) against tonic
convulsions induced by maximal electroshock (MES) according
to published methods.11 Compounds were injected intraperi-
toneally (ip) 30 min before induction of seizures. In this test,
compound (-)-10i displayed an ED50 of 4.5 ( 0.5 mg/kg (eight
runs, 12% variability). The other compounds were tested once
only.
In Vitr o Electr op h ysiology. Functional responses medi-
ated by NMDA receptors were obtained using the whole-cell
configuration of the patch-clamp technique on cerebellar
neurons cultured from 7-day-old Sprague-Dawley rat pups.
NMDA (50 µM) was applied for 5 s pulses using a rapid micro-
perfusion technique while glycine (3 µM) was added in the
bathing medium. Under these conditions, NMDA application
produced a brief peak of inward current that decayed rapidly
to a steady level. To obtain reliable measurements of current
amplitude, the mean current level during the NMDA applica-
tion was calculated and used to evaluate the effects of
antagonists. The effects of (-)-10i (0.1-10 nM) were evalu-
ated, after a preincubation period of 5 min in the bathing
medium, by application of a mixture of the two compounds
(NMDA + (-)-10i).
In Vivo Electr op h ysiology. Long-term potentiation (LTP)
was recorded in the hippocampus of intact, urethane-anaes-
thetized Sprague-Dawley rats. Excitatory postsynaptic po-
tential (epsp) were recorded extracellularly in the hippocampus
(CA1 area) using appropriate waveform processing and data
analysis software. For synaptic strength measurement, single
stimulations (0.1 ms duration) were delivered every 30 s at a
voltage giving approximately half the maximal response. LTP
was induced by applying three sets of high-frequency stimula-
tions, each set consisting of 10 trains of 50 stimuli at 4 ms
interval ()250 Hz). The intertrain interval was 10 s and the
interset interval was 5 min. (-)-10i was given intravenously
[3H]5,7-Dich lor ok yn u r en ic Acid Bin d in g. Membranes
from rat cerebral cortex were prepared according to a pub-
lished method.17 Briefly, rats were decapitated and their
cerebral cortices removed on ice and frozen at -80 °C for at
least 1 h. The tissue was rapidly thawed, homogenized with a
Polytron in 10 volumes of cold (4 °C) sucrose (0.32 M), and
centrifuged at 1000g for 10 min. The supernatant was recov-
ered and recentrifuged at 20000g for 20 min. The resulting
pellet was resuspended in 20 volumes of ice-cold distilled water
and centrifuged at 8000g for 20 min. The supernatant and
buffy layer were collected and centrifuged at 48000g for 20
min. The pellet was resuspended in 20 volumes of ice-cold
distilled water and recentrifuged at 48000g for 20 min. The
final pellet was frozen at -20 °C until use. On the day of the
binding assay, the membranes were thawed and resuspended
in 20 volumes of HEPES-KOH buffer (50 mM, pH 7.5),
incubated at 37 °C for 20 min, and then recentrifuged at
48000g for 10 min. This procedure was repeated once more.
The final pellet was resuspended in the appropriate buffer for
use in the binding assay. The extent of [3H]5,7-dichlo-
rokynurenic acid binding to the glycine recognition site on the
NMDA receptor was determined by the method described
previously.10 Briefly, membranes were suspended in HEPES-
KOH buffer (0.1 mg of protein/mL) and incubated for 10 min
at 4 °C with [3H]5,7-dichlorokynurenic acid (20 nM), the
studied compound, or 1 mM glycine for determination of the
nonspecific binding. The binding interaction was terminated

Indeno[1,2-b]pyrazin-2,3-diones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2381
Stutzmann, J . M.; Mignani, S. Synthesis and SAR of 2H-1,2,4-
Benzothiadiazine-1,1-dioxide-3-carboxylic acid Derivatives as
Novel Potent Glycine Antagonists of the NMDA Receptor-
Channel Complex. Bioorg. Med. Chem. Lett. 1994, 4, 2735-2740.
(b) Boireau, A.; Malgouris, C.; Burgevin, M. C.; Peny, C.; Durand,
G.; Bordier, F.; Miquet, J . M.; Meunier, M.; Daniel, M.; Chevet,
T.; J imonet, P.; Mignani, S.; Blanchard, J . C.; Doble, A. Neuro-
protective Effects of RPR 104632, a Novel Antagonist at the
Glycine Site of the NMDA Receptor, in vitro. Eur. J . Pharmacol.
1996, 300, 237-246. (c) Mignani, S.; Aloup, J . C.; Barreau, M.;
Birraux, G.; Blanchard, J . C.; Bohme, A.; Boireau, A.; Damour,
D.; Doble, A.; J imonet, P.; Malgouris, C.; Mary, V.; Randle, J .
C. R.; Rataud, J . and Stutzmann, J . M. 2H-3,4-Dihydro-1,2,4-
via an intravenous catheter in saline under a volume of 5 mL/
kg after 30 min of stable baseline recording.
Ackn owledgm en t. We thank He´le`ne Bocquel, Fran-
c¸oise Bordier, Dominique Briet, Eric Brohan, Philippe
Hubert, Andre´ Madoux, Colette Pe´ny, and Michel Roux
for their technical assistance, Marc Vuilhorgne and his
team for analytical support, and Christopher J . Burns
for valuable discussions.
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