Synthesis, glycine/NMDA and AMPA binding activity of some new 2,5,6- trioxopyrazino[1,2,3-de]quinoxalines and of their restricted analogs 2,5- dioxo- and 4,5-dioxoimidazo[1,5,4-de]quinoxalines

Article abstract of DOI:10.1002/(SICI)1521-4184(19996)332:6<201::AID-ARDP201>3.0.CO;2-S

The synthesis of some new 1,2,3,5,6,7-hexahydro-2,5,6- trioxopyrazino[1,2,3-de]quinoxalines 1c-g and of their restricted analogs 2,4,5,6-tetrahydro-2,5-dioxo-1H- 2a-g and 5,6-dihydro-4,5-dioxo-4H- imidazo[1,5,4-de]quinoxalines 3a-d is reported. Compounds 1c-g, 2a-g, and 3a- d were tested for their binding activity at the glycine/NMDA and AMPA receptors. The results show that only the 6,6,6-tricyclic derivatives 1c-g are able to bind to the glycine/NMDA and AMPA receptors, although with lower affinity than the previously reported lead compounds 1a-b. In contrast, the 5,6,6-tricyclic derivatives 2a-g are inactive at both receptors and only one 4,5-dioxoimidazoquinoxaline (3b) displays a weak glycine/NMDA receptor affinity.

Full text of DOI:10.1002/(SICI)1521-4184(19996)332:6<201::AID-ARDP201>3.0.CO;2-S

New 2,5,6-Trioxopyrazino[1,2,3-de]quinoxalines
201
Synthesis, Glycine/NMDA and AMPA Binding Activity of Some New
2,5,6-Trioxopyrazino[1,2,3-de]quinoxalines and of Their Restricted
Analogs 2,5-Dioxo- and 4,5-Dioxoimidazo[1,5,4-de]quinoxalines
a)
a)
a)
a)
a)
b)
Flavia Varano , Daniela Catarzi , Vittoria Colotta , Lucia Cecchi *, Guido Filacchioni , Alessandro Galli , and
b)
Chiara Costagli
a) Dipartimento di Scienze Farmaceutiche, Università di Firenze, Via Gino Capponi, 9, 50121 Firenze, Italy
b) Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Viale Pieraccini,6, 50134 Firenze, Italy
Key Words: 2,5,6-Trioxopyrazino[1,2,3-de]quinoxalines; 2,5-dioxoimidazo[1,5,4-de]quinoxalines;
4,5-dioxoimidazo[1,5,4-de]quinoxalines; glycine/NMDA and AMPA binding activity
Summary
The synthesis of some new 1,2,3,5,6,7-hexahydro-2,5,6-tri-
oxopyrazino[1,2,3-de]quinoxalines 1c–g and of their restricted
analogs 2,4,5,6-tetrahydro-2,5-dioxo-1H- 2a–g and 5,6-dihydro-
4,5-dioxo-4H-imidazo[1,5,4-de]quinoxalines 3a–d is reported.
Compounds 1c–g, 2a–g, and 3a–d were tested for their binding
activity at the glycine/NMDA and AMPA receptors. The results
show that only the 6,6,6-tricyclic derivatives 1c–g are able to bind
to the glycine/NMDA and AMPA receptors, although with lower
affinity than the previously reported lead compounds 1a–b. In
contrast, the 5,6,6-tricyclic derivatives 2a–g are inactive at both
receptors and only one 4,5-dioxoimidazoquinoxaline (3b) displays
a weak glycine/NMDA receptor affinity.
Introduction
Chart 1
NMDA and AMPA receptor subtypes are the best studied
among the ionotropic glutamate receptors. AMPA receptors
are responsible for most of the fast excitatory synaptic re-
sponses involved in neuronal transmission, while NMDA
imidazo[1,5,4-de]quinoxaline-4,5-diones 3a–d. Compounds
1c–g, 2a–g and 3a–d, together with the previously reported
1a–b were tested for their ability to displace radiolabeled
glycine and AMPA from their specific binding in rat cerebral
membranes. The synthesis of the 5,6,6-tricyclic 2,5-diones
2a–g and 4,5-diones 3a–d was performed to assess the im-
portance of the quinoxalinedione and pyrazinone moieties,
respectively, in the anchoring of the parent 2,5,6-trioxopyraz-
inoquinoxalines 1a–g to the glycine/NMDA and/or AMPA
recognition sites.
receptors are associated with slow, long-lasting currents
[1–2]
which mediate neuronal plasticity and activity
. Over-
stimulation of both these receptors by excessive endogenous
[3]
glutamate occurs in several pathological conditions
.
The NMDA receptor, like other ligand-gated ion channels,
is modulated by a number of molecules acting at different
allosteric sites and the amino acid glycine (glycine/NMDA)
is particularly important among them for its unique glutamate
[4]
co-agonist action in channel gating . Antagonists at both
glycine/NMDA and AMPA receptors are of great interest for
Chemistry
[5–6]
their potential therapeutic use
. Compounds with com-
bined glycine/NMDA and AMPA receptor antagonist activi-
The synthesis of compounds 1c–g, 2a–g, and 3a–d is shown
[7–8]
ties have been reported
, leading to the identification of in Schemes 1–3. Briefly, by reacting the 2,6-dinitrochlo-
[11]
the common structural requirements for receptor-ligand in- robenzene
with (±) phenylglycine or (±) glutamic acid
[3, 6, 9]
[10]
teraction
As a part of a program aimed at finding new antagonists of N-(2,6-dinitrophenyl)phenylglycine 5 and N-(2,6-dinitro-
the glycine/NMDA binding site, we recently reported the
synthesis and glycine/NMDA binding activity of ethyl (±) 1). Compounds 4–6 were transformed into the corresponding
.
following the method described to prepare 4
, the racemic
phenyl)glutamic acid 6 were obtained, respectively (Scheme
[10]
1,2,3,5,6,7-hexahydro-2,5,6-trioxopyrazino[1,2,3-de]quino
esters 7a
, c–d which by double catalytic reduction (Pd/C
[10]
xalin-3-acetate 1a and its corresponding acid 1b
and then Ni/Raney) yielded the 3-substituted-quinoxalines8a
[10]
(Chart 1). In the present paper we report the synthesis of other
pyrazinoquinoxaline derivatives 1c–g and of their restricted described for the preparation of 1a
, c–d. The latter, by reaction with oxalyl chloride, as
[10]
, yielded the pyrazino-
analogs imidazo[1,5,4-de]quinoxaline-2,5-diones 2a–g and quinoxaline-2,5,6-triones 1c–d. Moreover, the reaction of
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/0606/0201 $17.50 +.50/0

202
Cecchi and co-workers
Scheme 3. Reagents: a) succinic anhydride, xylene. b) to prepare 10a: conc
H2SO4, EtOH; to prepare 10b: PhNH2, 1-hydroxy-1H-benzotriazole, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; to prepare 10c:
K2CO3, C6H5-CH2Cl. c) to prepare 11a–b: H2/Pd/C; to prepare 11c: TiCl3.
d) (COCl)2, Et3N. e) H2/Pd/C.
the 4-nitrobenzimidazole-2-propionic acid 9 was prepared.
The acid 9 was transformed into the ester 10a which was
catalytically reduced to the 4-amino ester 11a. The latter was
cyclized with oxalyl chloride to the ethyl 4,5-dioxoimid-
azo[1,5,4-de]quinoxaline-2-propionate 3a. The instability in
acidic medium of the ester 3a prevented its use to obtain other
derivatives. Thus, the key intermediate 9 was reacted with
aniline by using a combination of 1-(dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride and 1-hydroxy-1H-ben-
zotriazole to obtain the 4-nitrocarboxyamide 10b. Catalytic
reduction of 10b yielded the 4-amino 11b which was cyclized
with oxalyl chloride to 3b. Finally, the 4-nitro acid 9 was
reacted with benzyl chloride to give 10c which was reduced
Scheme 1. Reagents: a) RCH(NH2)COOH, NaHCO3. b) conc H2SO4, EtOH.
c) H2/Pd/C, conc HCl. d) H2/Ni-Raney. e) (COCl)2, Et3N. f) (Cl3CO)2CO,
Et3N.
with TiCl to 11c. Cyclization of 11c yielded 3c. The latter
3
was catalytically reduced to afford the 4,5-dioxoimid-
azo[1,5,4-de]quinoxaline-2-propionic acid 3d.
Results and Conclusions
The ability of compounds 1c–g, 2a–g and 3a–d to displace
radiolabeled glycine and AMPA from their specific binding
in rat brain synaptic membrane preparation, expressed as K
i
values, is listed in Table 1. The K values at the glycine/
i
NMDA and AMPA receptors of the previously reported
[10]
1a–b
have also been calculated and included in Table 1
together with the IC values at these same receptors of the
50
well known antagonist PNQX (1,4,7,8,9,10-hexahydro-9-
[6]
methyl-6-nitropyrido[3,4-f]quinoxalin-2,3-dione ).
Scheme 2. Reagents: a) 1M NaOH. b) PhNH2, 1-hydroxy-1H-benzotriazole,
1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride.
The results show that only the newly reported 2,5,6-tri-
oxopyrazino-quinoxalines 1c–g display some glycine/
NMDA and AMPA receptor affinities. In fact, the 2,5-di-
oxoimidazo-quinoxalines 2a–g are completely inactive at
both receptors, while only one 4,5-dioxoimidazoquinoxaline,
i.e. 3b, displays a weak affinity for the glycine/NMDA recep-
tor.
However, the newly prepared 6,6,6-tricyclic derivatives
1c–g show a significant loss of affinity at the glycine/NMDA
receptor with respect to that of the previously reported 1a. In
fact, the acetate side chain at position-3 of 1a seems to be the
8a, c–d with triphosgene to prepare the imidazoquinoxaline-
2,5-diones 2a, c–d is also illustrated in Scheme 1.
The pyrazinoquinoxaline esters 1a, d and imidazoquinox-
aline esters 2a, d were hydrolyzed to the corresponding acids
1b, e and 2b, e which were transformed into the carboxy-
amides 1f–g and 2f–g (Scheme 2).
Scheme 3 describes the synthetic pathway which yielded
the imidazoquinoxaline-4,5-diones 3a–d. Allowing the 3-ni-
trophenylene-1,2-diamine to react with succinic anhydride, best one and all changes such as its homologation (1d) or
Arch. Pharm. Pharm. Med. Chem. 332, 201–207 (1999)

New 2,5,6-Trioxopyrazino[1,2,3-de]quinoxalines
203
Table 1. Binding activity of tricyclic compounds.
In conclusion, the synthesis of the 6,6,6-tricyclic deriva-
tives 1c–g and of their restricted analogs 2a–g and 3a–d have
led to some weak glycine/NMDA and AMPA receptor li-
gands, although the binding results do not encourage further
investigations of these tricyclic systems.
Acknowledgement
Valuable assistance in animal handling was provided by M.G. Giovannini.
——————————————————————————————
Comp
——————————————————————————————
R
[3H]Gly Ki (µM)a
[3H]AMPA Ki(µM)a
Experimental Section
1a
CH2COOEt
CH2COOH
C6H5
2.3 ± 0.7
25.6 ± 1.8
> 100
> 100
Chemistry
1b
1c
49.7 ± 9.3
> 100
Silica gel plates (Merck F254) and silica gel 60 (Merck; 70–230 mesh) were
used for analytical and column chromatography, respectively. All melting
points were determined on a Gallenkamp melting point apparatus. Micro-
analyses were performed with a Perkin-Elmer 260 elemental analyzer for C,
H, N, and the results were within ± 0.4% of the theoretical values. The 1H
NMR spectra were obtained with a Varian Gemini 200 instrument at
200 MHz. The chemical shifts are reported in δ (ppm) and are relative to the
central peak of the solvent. The following abbreviations are used: s = singlet,
d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet, br =
broad, and ar = aromatic protons. Physical and 1H NMR data for the newly
synthesized compounds are listed in Tables 2–3.
1d
1e
(CH2)2COOEt
(CH2)2COOH
CH2CONH C6H5
> 100
> 100
37.1 ± 11.5
58.2 ± 15
19.6 ± 2.4
65 ± 7.6
> 100
1f
1g
(CH2)2CONH C6H5 88 ± 7.1
2a
CH2COOEt
CH2COOH
C6H5
> 100
> 100
> 100
> 100
> 100
> 100
> 100
2b
2c
> 100
> 100
2d
2e
(CH2)2COOEt
(CH2)2COOH
CH2CONH C6H5
> 100
(±) N-(2,6-Dinitrophenyl)phenylglycine 5 and (±) N-(2,6-Dinitrophenyl)glu-
tamic Acid 6
> 100
2f
> 100
The title compounds were obtained from the corresponding amino acid
(13.2 mmol) and 2,6-dinitrochlorobenzene (12 mmol) as described for the
2g
(CH2)2CONH C6H5 > 100
> 100
synthesis of 4 [10]
.
3a
OEt
> 100
61.8 ± 13
> 100
> 100
0.37
> 100
3b
3c
NH C6H5
OCH2 C6H5
OH
> 100
Ethyl (±) N-(2,6-Dinitrophenyl)phenylglycinate 7c and Diethyl (±) N-(2,6-
Dinitrophenyl)glutamate 7d
> 100
3d
PNQXb
> 100
The title compounds were obtained from 5–6 (7.4 mmol) as described for
the synthesis of 7a[10]
.
0.063 ± 0.012
——————————————————————————————
aThe values are ± SEM of 3–4 separate determinations in triplicate.
The tested compounds were dissolved in DMSO.
(±) 1,2,3,4-Tetrahydro-5-amino-3-phenylquinoxalin-2-one 8c and Ethyl (±)
1,2,3,4-Tetrahydro-5-amino-2-oxoquinoxaline-3-propionate 8d
Conc HCl (1.2 mL) and Pd/C (10%, 0.3 g) were added to a solution of 7c–d
(7.55 mmol) in absolute EtOH (200 mL). The mixture was hydrogenated in
a Parr apparatus at 30 psi for 12 h. Elimination of the catalyst and evaporation
at room temperature and reduced pressure of the solvent yielded a residue
which was dissolved in H2O (70 mL). The solution was neutralized with a
saturated solution of NaHCO3 and then extracted with ethyl acetate
(3 × 40 mL).The organic layers were dried (Na2SO4) and evaporated at room
temperature and reduced pressure to yield an oil which was dissolved in
EtOH (350 mL). Ni/Raney (50% slurry in H2O, 1 g) was added to the
ethanolic solution which was then hydrogenated in a Parr apparatus at 35 psi
for 24 h. Elimination of the catalyst and evaporation at room temperature and
reduced pressure of the solvent yielded a residue which became solid with
Et2O.
bIC50 values from Ref. 6.
replacement with a phenyl ring (1c) resulted in a significant
loss of glycine/NMDA receptor affinity. Moreover, in the
3-acetate series 1a–b, f, the ester 1a is active only at the
glycine/NMDA receptor, while the acid 1b and the anilide 1f
show a weak affinity at both receptors. It is however intrigu-
ing to note that in the 3-propionate series 1d–e, g, the ester
1d and the anilide 1g are devoid of binding activity at both
receptors while the acid 1e displays some glycine/NMDA and
AMPA receptor affinity.
The inactivity at both receptor sites of the five-membered
fused-ring 2,5-dioxoimidazo-quinoxalines 2a–g may be at-
(±) 1,2,3,5,6,7-Hexahydro-3-phenylpyrazino[1,2,3-de]quinoxaline-2,5,6-
trione 1c and Ethyl (±) 1,2,3,5,6,7-Hexahydro-2,5,6-trioxopyrazino[1,2,3-
de]quinoxaline-3-propionate 1d
tributed to the lack of the crucial quinoxalinedione moi-
[6,8]
ety
. The binding results of 5,6,6-tricyclic-4,5-dioxo-
The title compounds were obtained from 8c–d (2 mmol) as described for
the synthesis of 1a [10]
.
imidazoquinoxalines 3a–d do not allow any conclusion about
the importance of the pyrazinone moiety. In fact, by compar-
ing the glycine/NMDA and AMPA binding activities of
compounds 3a–b, d with those of their enlarged analogs 1d,
1g, and 1e, the similar affinity trend of the esters 3a and 1d
and that of the anilides 3b and 1g contrasts with the inactivity
of the acid 3d with respect to the activity of its corresponding
propionic acid 1e.
(±) Ethyl 2,4,5,6-Tetrahydro-2,5-dioxo-1H-imidazo[1,5,4-de]quinoxalin-4-
acetate 2a, (±) 2,4,5,6-Tetrahydro-4-phenyl-1H-imidazo[1,5,4-de]quinox-
aline-2,5-dione 2c and (±) Ethyl 2,4,5,6-Tetrahydro-2,5-dioxo-
1H-imidazo[1,5,4-de]quinoxaline-4-propionate 2d
Triphosgene (1.8 mmol) and triethylamine (9 mmol) were added to a
solution of 8a, c–d (4.5 mmol) in anhydrous tetrahydrofuran (THF)
Arch. Pharm. Pharm. Med. Chem. 332, 201–207 (1999)

204
Cecchi and co-workers
(40 mL).The mixture was stirred at room temperature and under nitrogen
atmosphere until disappearance of the starting material (TLC monitoring).
Dilution with H2O (30 mL) afforded a solid which was collected and washed
with Et2O.
(±) 1,2,3,5,6,7-Hexahydro-2,5,6-trioxopyrazino[1,2,3-de]quinoxaline-3-
propionic Acid 1e
The title compound was obtained by hydrolysis of 1d (0.66 mmol) as
described to prepare 1b [10]
.
Table 2. Physical and 1H NMR data of the intermediates.
—————————————————————————————————————————————————————————————–
Solva 1H NMR (DMSO-d6)
Comp
Mp(°C)
Yield(%)
—————————————————————————————————————————————————————————————–
5
oil
78
5.05 (d, 1H, CH, J = 6.0 Hz), 6.93 (t, 1H, ar, J = 8.2 Hz), 7.11–7.29 (m, 5H, ar), 8.23 (d, 2H, ar,
J = 8.2 Hz), 9.57 (d, 1H, NH, J = 6.0 Hz).
6
oil
80
1.95–2.03 (m, 2H, CH2), 2.25–2.32 (m, 2H, CH2), 3.77–3.88 (m, 1H, CH), 7.07 (t, 1H, ar,
J = 8.2 Hz), 7.99 (d, 1H, NH, J = 9.4 Hz), 8.30 (d, 2H, ar, J = 8.2 Hz), 12.45 (br s, exchangeble
protons).
7c
oil
oil
75
87
1.10 (t, 3H, CH3, J = 7.1 Hz), 4.15 (q, 2H, CH2, J = 7.1 Hz), 5.14 (d, 1H, CH, J = 6.4 Hz),
6.98 (t, 1H, ar, J = 8.2 Hz), 7.16–7.33 (m, 5H, ar), 8.27 (d, 2H, ar, J = 8.2 Hz), 9.41 (d, 1H, NH,
J = 6.4 Hz).
7d
1.09–1.20 (m, 6H, 2 CH3), 1.99–2.10 (m, 2H, CH2), 2.38–2.45 (m, 2H, CH2), 3.78–3.89 (m, 1H, CH),
3.97–4.14 (m, 4H, 2CH2 esters), 7.11 (t, 1H, ar, J = 8.2 Hz), 7.73 (d, 1H, NH, J = 9.8 Hz),
8.29 (d, 2H, ar, J = 8.2 Hz).
8c
210–213
87–89
A
A
50
60
4.78–4.90 (m, 3H, H-3 + NH2), 5.65 (s, 1H, NH), 6.10 (d, 1H, ar, J = 7.3 Hz), 6.26 (d, 1H, ar,
J = 7.3 Hz), 6.43 (t, 1H, ar, J = 7.3 Hz), 7.27 (s, 5H, ar), 10.21 (s, 1H, NH).
8d
1.18 (t, 3H, CH3, J = 7.0 Hz), 1.76–1.93 (m, 2H, CH2), 2.43–2.47 (m, 2H, CH2), 3.67–3.70 (m,
1H, H-3), 4.06 (q, 2H, CH2, J = 7.0 Hz), 4.76 (s, 2H, NH2), 4.99 (d, 1H, NH, J = 2.4 Hz),
6.11 (d, 1H, ar, J = 7.6 Hz), 6.24 (d, 1H, ar, J = 7.6 Hz), 6.43 (t, 1H, ar, J = 7.6 Hz), 10.07 (s, 1H, NH).
9
246–247
172–174
222–224
174–176
83–85
B
C
D
D
E
F
75
90
70
58
75
88
75
2.85 (t, 2H, CH2, J = 7.1 Hz), 3.18 (t, 2H, CH2, J = 7.1 Hz), 7.35 (t, 1H, ar, J = 8.1 Hz),
8.03 (d, 1H, ar, J = 8.1 Hz), 8.07 (d, 1H, ar, J = 8.1 Hz), 12.30 (br s, 1H, NH or OH),
13.00 (br s, 1H, OH or NH).
10a
10b
10c
11a
11b
11c
1.17 (t, 3H, CH3, J = 7.0 Hz), 2.92 (t, 2H, CH2, J = 7.1 Hz), 3.22 (t, 2H, CH2, J = 7.1 Hz),
4.07 (q, 2H, CH2, J = 7.0 Hz), 7.36 (t, 1H, ar, J = 8.1 Hz), 8.02 (d, 1H, ar, J = 8.1 Hz),
8.08 (d, 1H, ar, J = 8.1 Hz), 13.07 (s, 1H, NH).
2.96 (t, 2H, CH2, J = 7.7 Hz), 3.26 (t, 2H, CH2, J = 7.7 Hz), 7.05 (t, 1H, ar, J = 7.7 Hz),
7.24–7.38 (m, 3H, ar), 7.59 (d, 2H, ar, J = 8.3 Hz), 7.99–8.10 (m, 2H, ar), 10.07 (s, 1H, NH),
13.08 (s, 1H, NH).
3.00 (t, 2H, CH2, J = 7.0 Hz), 3.25 (t, 2H, CH2, J = 7.0 Hz), 5.10 (s, 2H, CH2), 7.29 ( s, 5H, ar),
7.37 (t, 1H, ar, J = 8.1 Hz), 8.02 (d, 1H, ar, J = 8.1 Hz), 8.08 (d, 1H, ar, J = 8.1 Hz),
13.15 (br s, 1H, NH).
1.18 (t, 3H, CH3, J = 7.1 Hz), 2.84 (t, 2H, CH2, J = 6.9 Hz), 2.96 (t, 2H, CH2, J = 6.9 Hz),
4.05 (q, 2H, CH2, J = 7.1 Hz), 5.04 (s, 2H, NH2), 6.29 (d, 1H, ar, J = 7.7 Hz),
6.60 (d, 1H, ar, J = 7.7 Hz), 6.81 (t, 1H, ar, J = 7.7 Hz), 11.89 (s, 1H, NH).
192–194
107–108
2.89 (t, 2H, CH2, J = 7.1 Hz), 3.10 (t, 2H, CH2, J = 7.1 Hz), 5.15 (br s, 2H, NH2),
6.33 (d, 1H, ar, J = 7.5 Hz), 6.66 (d, 1H, ar, J = 7.7 Hz), 6.82 (t, 1H, ar, J = 7.5 Hz),
7.03 (t, 1H, ar, J = 7.5 Hz), 7.30 (t, 2H, ar, J = 7.7 Hz), 7.61 (d, 2H, ar, J = 7.7 Hz), 10.08 (s, 1H, NH).
E
2.94 (t, 2H, CH2, J = 6.0 Hz), 3.03 (t, 2H, CH2, J = 6.0 Hz), 5.04 (s, 2H, NH2), 5.12 (s, 2H, CH2),
6.28 (d, 1H, ar, J = 7.7 Hz), 6.60 (d, 1H, ar, J = 7.7 Hz), 6.82 (t, 1H, ar, J = 7.7 Hz), 7.32 (s, 5H, ar),
11.91 (s, 1H, NH).
—————————————————————————————————————————————————————————————–
aPurification solvents: A = Benzene. B = Glacial acetic acid. C = Ethyl acetate. D = Ethanol. E = Diethyl ether. F = Acetonitrile.
Arch. Pharm. Pharm. Med. Chem. 332, 201–207 (1999)

New 2,5,6-Trioxopyrazino[1,2,3-de]quinoxalines
205
Table 3. Physical and 1H NMR data of the final tricyclic compounds.
—————————————————————————————————————————————————————————————–
Solva 1H NMR (DMSO-d6)
Comp
Mp(°C)
Yield(%)
—————————————————————————————————————————————————————————————–
1c
268–272
A
30
5.95 (s, 1H, H-3), 6.78 (d, 1H, ar, J = 8.0 Hz), 6.88 (d, 1H, ar, J = 8.0 Hz), 7.12–7.34 (m, 6H, ar),
11.14 (s, 1H, NH), 12.25 (s, 1H, NH).
1d
248–251
B
45
1.07 (t, 3H, CH3, J = 7.2 Hz), 2.04–2.36 (m, 4H, 2CH2), 3.86–3.91 (m, 2H, CH2 esters),
5.03–5.08 (m, 1H, H-3), 6.69 (d, 1H, ar, J = 7.7 Hz), 6.79 (d, 1H, ar, J = 7.7 Hz), 7.08 (t, 1H, ar,
J = 7.7 Hz), 11.09 (s, 1H, NH), 12.07 (s, 1H, NH).
1e
1f
> 300
A
C
D
E
80
60
50
75
2.02–2.27 (m, 4H, 2CH2), 5.02–5.07 (m, 1H, H-3), 6.69 (d, 1H, ar, J = 8.2 Hz), 6.78 (d, 1H, ar,
J = 8.2 Hz), 7.07 (t, 1H, ar, J = 8.2 Hz), 11.10 (s, 1H, NH), 12.15 (br s, 2H, NH + OH).
> 300
3.09–3.32 (m, 2H, CH2), 5.15–5.21 (m, 1H, H-3), 6.67–6.77 (m, 2H, ar), 6.98–7.09 (m, 2H, ar),
7.17–7.35 (m, 4H, ar), 10.01 (s, 1H, NH), 11.00 (s, 1H, NH), 12.11 (s, 1H, NH).
1g
2a
265–267
246–248
2.15–2.32 (m, 4H, 2CH2), 5.03–5.15 (m, 1H, H-3), 6.67–6.75 (m, 2H, ar), 6.98–7.08 (m, 2H, ar),
7.19–7.26 (m, 2H, ar), 7.42–7.46 (m, 2H, ar), 9.77 (s, 1H, NH), 11.06 (s, 1H, NH), 12.15 (s, 1H, NH).
1.01 (t, 3H, CH3, J = 7.0 Hz), 3.21 (dd, 1H, CH2, J = 17.2, 3.0 Hz), 3.48 (dd, 1H, CH2,
J = 17.2, 4.6 Hz), 3.94 (q, 2H, CH2, J = 7.0 Hz), 5.02–5.05 (m, 1H, H-4), 6.48 (d, 1H, ar, J = 7.7 Hz),
6.62 (d, 1H, ar, J = 7.7 Hz), 6.86 (t, 1H, ar, J = 7.7 Hz), 10.79 (s, 1H, NH), 10.85 (s, 1H, NH).
2b
279–282
D
80
3.15 (dd, 1H, CH2, J = 17.5, 3.0 Hz), 3.47 (dd, 1H, CH2, J = 17.5, 4.2 Hz), 4.95–4.98 (m, 1H, H-4),
6.46 (d, 1H, ar, J = 7.9 Hz), 6.61 (d, 1H, ar, J = 7.9 Hz), 6.85 (t, 1H, ar, J = 7.9 Hz), 10.72
(s, 1H, NH), 10.84 (s, 1H, NH), 12.40 (s, 1H, OH).
2c
> 300
D
E
65
70
5.77 (s, 1H, H-4), 6.57 (d, 1H, ar, J = 7.8 Hz), 6.71 (d, 1H, ar, J = 7.8 Hz), 6.94 (t, 1H, ar, J = 7.8 Hz),
7.15–7.19 (m, 2H, ar), 7.33–7.38 (m, 3H, ar), 10.90 (s, 1H, NH), 10.94 (s, 1H, NH).
2d
212–214
1.07 (t, 3H, CH3, J = 7.1 Hz), 2.19–2.36 (m, 4H, 2CH2), 3.83–3.96 (m, 2H, CH2 esters),
4.89–4.92 (m, 1H, H-4), 6.47 (d, 1H, ar, J = 7.9 Hz), 6.62 (d, 1H, ar, J = 7.9 Hz),
6.86 (t, 1H, ar, J = 7.9 Hz), 10.82 (s, 1H, NH), 10.90 (s, 1H, NH).
2e
2f
> 300
> 300
A
D
75
60
2.11–2.46 (m, 4H, 2CH2), 4.88–4.91 (m, 1H, H-4), 6.47 (d, 1H, ar, J = 7.8 Hz),
6.62 (d, 1H, ar, J = 7.8 Hz), 6.86 (t, 1H, ar, J = 7.8 Hz), 10.82 (s, 1H, NH), 10.94 (s, 1H, NH),
12.15 (s, 1H, OH).
3.26 (dd, 1H, CH2, J = 16.5, 2.9 Hz), 3.69 (dd, 1H, CH2, J = 16.5, 4.0 Hz), 4.96–5.09 (m, 1H, H-4),
6.50 (d, 1H, ar, J = 7.9 Hz), 6.61 (d, 1H, ar, J = 7.9 Hz), 6.83–7.03 (m, 2H, ar),
7.22 (t, 2H, ar, J = 7.9 Hz), 7.41 (d, 2H, ar, J = 7.9 Hz), 10.09 (s, 1H, NH), 10.70 (s, 1H, NH),
10.77 (s, 1H, NH).
2g
3a
287–289
206–207
D
F
70
35
2.10–2.47 (m, 4H, 2CH2), 4.91–4.99 (m, 1H, H-4), 6.48 (d, 1H, ar, J = 7.7 Hz), 6.64 (d, 1H, ar,
J = 7.7 Hz), 6.84 (t, 1H, ar, J = 7.7 Hz), 7.01 (m, 1H, ar), 7.26 (t, 2H, ar, J = 7.7 Hz),
7.51 (d, 2H, ar, J = 7.7 Hz), 9.87 (s, 1H, NH), 10.86 (s, 1H, NH), 10.92 (s, 1H, NH).
1.21 (t, 3H, CH3, J = 7.1 Hz), 2.94 (t, 2H, CH2, J = 7.0 Hz), 3.49 (t, 2H, CH2, J = 7.0 Hz),
4.11 (q, 2H, CH2, J = 7.1 Hz), 7.01 (d, 1H, ar, J = 7.6 Hz), 7.32 (d, 1H, ar, J = 7.6 Hz),
7.42 (t, 1H, ar, J = 7.6 Hz), 12.02 (s, 1H, NH).
3b
3c
3d
247–250
212–213
233–234
B
F
F
38
30
60
2.95–3.03 (m, 2H, CH2), 3.49–3.58 (m, 2H, CH2), 6.99–7.07 (m, 2H, ar), 7.22–7.44 (m, 4H, ar),
7.57–7.64 (m, 2H, ar), 10.08 (s, 1H, NH), 12.03 (s, 1H, NH).
3.03 (t, 2H, CH2, J = 6.9 Hz), 3.52 (t, 2H, CH2, J = 6.9 Hz), 5.12 (s, 2H, CH2),
7.01 (d, 1H, ar, J = 7.5 Hz), 7.30–7.46 (m, 7H, ar), 12.02 (s, 1H, NH).
2.86 (t, 2H, CH2, J = 7.1 Hz), 3.44 (t, 2H, CH2, J = 7.1 Hz), 7.01 (d, 1H, ar, J = 7.5 Hz),
7.29–7.45 (m, 2H, ar), 11.99 (s, 1H, NH or OH), 12.30 (brs, 1H, OH or NH).
—————————————————————————————————————————————————————————————–
aPurification solvents: A = Dimethylformamide/water. B = Nitromethane. C = Dimethylformamide. D = Glacial acetic acid. E = Ethanol.
F = Ethyl acetate.
Arch. Pharm. Pharm. Med. Chem. 332, 201–207 (1999)

206
Cecchi and co-workers
(±)1,2,3,5,6,7-Hexahydro-2,5,6-trioxopyrazino[1,2,3-de]quinoxaline-3-N-
phenylacetamide 1f and (±) 1,2,3,5,6,7-Hexahydro-2,5,6-trioxopyraz-
ino[1,2,3-de]quinoxaline-3-N-phenylpropionamide 1g
ganic layers were washed with H2O (3 × 20 mL) and dried (Na2SO4). Evapo-
ration of the solvent at reducedpressureyieldedanoilyresiduewhichbecame
solid upon treatment with petroleum ether (40–60 °C) .
Equimolar amount (0.68 mmol) of aniline, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride and 1-hydroxy-1H-benzotriazole were
added to a solution of acid 1b, e (0.62 mmol) in anhydrous dimethylfor-
mamide (DMF) (3 mL). The mixture was stirred at room temperature for
20 h. Addition of HCl (0.1 M, 10 mL) yielded a solid which was collected
and washed with H2O.
4-Aminobenzimidazole-2-N-phenylpropionamide 11b
Pd/C (10%, 0.1 g) was added to a hot solution of 10b (1.2 mmol) in
absolute EtOH (100 mL). The mixture was hydrogenated in a Parr apparatus
at 40 psi for 3 h. Elimination of the catalyst and evaporation of the solvent
at reduced pressure yielded a residue which became solid with Et2O.
(±) 2,4,5,6-Tetrahydro-2,5-dioxo-1H-imidazo[1,5,4-de]quinoxalin-4-acetic
Acid 2b and (±) 2,4,5,6-Tetrahydro-2,5-dioxo-1H-imidazo[1,5,4-de]quinox-
aline-4-propionic Acid 2e
Benzyl 4-Aminobenzimidazole-2-propionate 11c.
A solution of 10c (1.63 mmol) in acetone (50 mL) was slowly (1 h) added
to a stirred and cooled (0 °C) solution of TiCl3 (20%, in H2O, 16.3 mmol) in
H2O (5 mL) and acetone (5 mL). The mixture was stirred and cooled for
30 min. The acetone was evaporated at reduced pressure and the aqueous
mixture was added with H2O (70 mL) and ethyl acetate (150 mL), and then
alkalinized with NaHCO3 (saturated solution). The mixture was stirred for
30 min. The solid was filtered off and the two layers were separated.
Evaporation at reduced pressure of the dried (Na2SO4) organic solvent
yielded an oily residue which became solid upon treatment with Et2O.
The title compounds were obtained by hydrolysis of the corresponding
esters 2a, d (2.96 mmol) as described for the synthesis of 1b [10]
.
(±) 2,4,5,6-Tetrahydro-2,5-dioxo-1H-imidazo[1,5,4-de]quinoxalin-4-N-
phenylacetamide 2f and (±) 2,4,5,6-Tetrahydro-2,5-dioxo-1H-imid-
azo[1,5,4-de]quinoxaline-4-N-phenylpropionamide 2g
The title compounds were obtained from the acids 2b, e (1.46 mmol) as
described above to prepare 1f–g.
Ethyl 5,6-Dihydro-4,5-dioxo-4H-imidazo[1,5,4-de]quinoxaline-2-propion-
ate 3a, 5,6-Dihydro-4,5-dioxo-4H-imidazo[1,5,4-de]quinoxaline-2-N-
phenylpropionamide 3b, and Benzyl 5,6-Dihydro-4,5-dioxo-4H-imid-
azo[1,5,4-de]quinoxaline-2-propionate 3c
4-Nitrobenzimidazole-2-propionic Acid 9
An excess of succinic anhydride (39.2 mmol) was added to a suspension
of 3-nitrophenylene-1,2-diamine (19.6 mmol) in xylene (100 mL). The mix-
ture was refluxed for 6 h. The crude acid 9, that resulted upon cooling, was
collected and purified as follows: the acid 9 was suspended in H2O (35 mL)
and the suspension was slightly alkalinized (pH ≈ 8) with NaOH (10%). The
solid was filtered off and the clear solution was acidified with glacial acetic
acid to yield a solid which was collected and washed with H2O.
A solution of 11a–c (2.14 mmol) and triethylamine (4.28 mmol) in THF
(20 mL) was slowly (3 h) added under nitrogen atmosphere to a stirred and
cooled (0 °C) solution of oxalyl chloride (2.35 mmol) in THF (20 mL). The
mixture was stirred and cooled for 30 min. Dilution with H2O (70 mL)
yielded compounds 3a–b as a milky suspension which was extracted with
ethyl acetate (3 × 40 mL). The organic layers were washed with H2O
(2 × 50 mL) and dried (Na2SO4). Evaporation of the solvent at reduced
pressure afforded a residue which was treated with Et2O and collected. In the
case of 3c the dilution with H2O gave a solid which was collected, washed
with H2O, and suspended in ethyl acetate (40 mL). The solid was filtered off
and the solution was concentrated ( 10 mL) to yield crude 3c.
Ethyl 4-Nitrobenzimidazole-2-propionate 10a
Conc H2SO4 (0.89 mL) was added to a solution of 9 (12.4 mmol) in
absolute EtOH (130 mL). The solution was refluxed for 3 h. Elimination of
the solvent at reduced pressure yielded a residue which was treated with H2O
(60 mL). The mixture was alkalinized with NaHCO3 (saturated solution) to
produce a solid which was collected and washed with H2O.
5,6-Dihydro-4,5-dioxo-4H-imidazo[1,5,4-de]quinoxaline-2-propionic Acid
3d
Pd/C (10%, 0.1 g) was added to a solution of 3c (0.51 mmol) in ethyl
acetate (200 mL). The mixture was hydrogenated in a Parr apparatus at 35 psi
for 2 h. Elimination of the catalyst and evaporation at reduced pressure of
the solvent yielded a residue which was washed with Et2O.
4-Nitrobenzimidazole-2-N-phenylpropionamide 10b
A solution of 9 (2.5 mmol) in anhydrous DMF (5 mL) was added dropwise
and under stirring to a cooled (0 °C) suspension of 1-hydroxy-1H-benzotri-
azole (5.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (5.1 mmol) and aniline (3 mmol) in anhydrous DMF. The mixture
was stirred at room temperature for 12 h. Dilution with H2O (15 mL) and
alkalization with NaHCO3 (saturated solution) yielded a solid which was
collected and washed with H2O.
Biochemistry
Synaptic Membrane Preparation
Crude synaptic membranes were prepared from the cerebral cortex plus
hippocampus of male Sprague-Dawley rats (170–250 g). The tissue was
homogenized in 20 vol of ice-cold 0.32 M sucrose, containing 20 µg/mL
phenylmethanesulfonyl fluoride, using a glass-Teflon homogenizer (clear-
ance = 0.15–0.23 mm). The homogenate was centrifuged at 1,000 × g for
10 min and the resulting supernatant further centrifuged at 20,000 × g for
20 min. The final pellet was resuspended in 20 vol of ice-cold distilled water,
dispersed with an Ultra-Turrax sonicator (30% of maximum speed) for 30 s
and centrifuged at 8,000 × g for 20 min. The supernatant and the soft upper
layer of the pellet were collected together and centrifuged at 48,000 × g for
20 min. The membranes were resuspended once more in distilled water,
centrifuged and frozen at –80 °C.
Benzyl 4-Nitrobenzimidazole-2-propionate 10c
K2CO3 (0.48 g) was added to a stirred solution of 9 (4.59 mmol) in
anhydrous DMF (60 mL). The mixture was stirred at room temperature for
4 h. Benzyl chloride (5.5 mmol) was added to the mixture which was stirred,
still at room temperature, for another 24 h. The mixture was then concen-
trated (30 mL) at reduced pressure and at 100 °C and diluted with H2O
(70 mL) to yield a solid which was collected, washed with H2O and purified
by column chromatography, eluting system CHCl3/ethyl acetate (9:1).
Evaporation of the central eluates gave a residue which was washed with
Et2O.
Ethyl 4-Aminobenzimidazole-2-propionate 11a
[3H]Glycine Binding
Conc HCl (0.25 mL) and Pd/C (10%, 0.4 g) were added to a solution of
10a (1.5 mmol) in absolute EtOH (40 mL). The mixture was hydrogenated
in a Parr apparatus at 40 psi for 2 h. Elimination of the catalyst and evapora-
tion at reduced pressure of the solvent yielded an oily residue which was
dissolved in H2O (15 mL). The solution was alkalinized with NaHCO3
(saturated solution) and extracted with ethyl acetate (3 × 30 mL). The or-
On the day of the experiment, appropriate amounts of membranes were
thawed at room temperature, resuspended (0.5 mg protein/mL) in cold
0.05 M Tris acetate buffer, pH 7.0, containing 0.08% v/v Triton X-100, and
stirred for 10 min at 0–2 °C. The membranes were then collected by centrifu-
gation at 48,000 × g for 10 min, submitted to four additional resuspension
Arch. Pharm. Pharm. Med. Chem. 332, 201–207 (1999)

New 2,5,6-Trioxopyrazino[1,2,3-de]quinoxalines
207
and centrifugation cycles with fresh buffer and finally resuspended in cold
Tris acetate buffer to yield 0.2–0.3 mg protein/assay tube. [3H]Glycine
(51.3 Ci/mmol) binding assays were carried out in ice for 30 min at 10 nM
ligand concentration in a total 1.0 mL vol. Bound radioactivity was separated
by rapid filtration through Whatman GF/B discs using a Millipore filtration
apparatus. Non-specific binding was determined in the presence of 1 mM
D-serine.
References
[1] D.K. Patneau, M.L. Mayer, Neuron 1991, 6, 785–798.
[2] I.D. Forsythe, G.L. Nestbrook, J. Physiol. Lond. 1988, 396, 515–534.
[3] P.D. Leeson, L.L. Iversen, J. Med. Chem. 1994, 37, 4053–4067.
[4] J. W. Johnson, P. Ascher, Nature 1987, 325, 529–531.
[5] J.L. Mosinger, M.T. Price, H.Y. Bai, H. Xiao, D.F. Wozniak, J.W.
[3H]DL-AMPA Binding
Olney, Exp. Neurol. 1991, 113, 10–17.
[6] C.F. Bigge, T.C. Malone, P.A. Boxer, C.B. Nelson, D.F. Ortwine, R.M.
Schelkun, D.M. Retz, L.J. Lescosky, S.A. Borosky, M.G. Vartanian,
R.D. Schwarz, G.W. Campbell, L.J. Robichaud, F. Watjen, J. Med.
Chem. 1995, 38, 3720–3740.
[3H]DL-AMPA binding assays were carried out basically following the
method of Nielsen et al.[12] Briefly, frozen membranes were resuspended in
cold 30 mM Tris-HCl buffer, pH 7.1, containing 2.5 mM CaCl2 and centri-
fuged at 48,000 × g for 10 min. After an additional washing step, the pellet
was resuspended (0.5–0.6 mg protein/mL) in the original buffer containing
2.5 mM CaCl2 and 100 mM KSCN and used for binding assays. Aliquots of
0.5 mL membrane suspensions in triplicate were incubated for 30 min at
0–2 °C with 5 nM [3H]DL-AMPA (40 Ci/mmol) in the absence or presence
of scalar concentrations of the test compound. Non-specific binding was
assessed in the presence of 10 µM 6-nitro-7-sulfamoylbenzo[f]quinoxaline-
2,3-dione (NBQX). Bound radioactivity was separated by filtration through
Whatman GF/C glass fibre discs followed by washing with 3 × 5 mL ice-cold
buffer.
[7] J.J. Kulagowski, Exp. Opin. Ther. Patents 1996, 6, 1069–1079.
[8] C.F. Bigge, S.S. Nikam, Exp. Opin. Ther. Patents 1997, 7, 1099–1114.
[9] R.W. Carling, P.D. Leeson, K.W. Moore, J.D. Smith, C.R. Moyes, I.W.
Mawer, S. Thomas, T. Chan, R. Baker, A.C. Foster, S. Grimwood, J.A.
Kemp, G.R. Marshall, M.D. Tricklebank, K.L. Saywood, J. Med.
Chem. 1993, 36, 3397–3408.
[10] F. Varano, D. Catarzi, V. Colotta, L. Cecchi, G. Filacchioni, A. Galli,
C. Costagli, Farmaco 1997, 52, 179–182.
[11] F.D. Gunston, S.H. Tucker, Org. Syn. 1963 Coll. Vol. IV, 160–161.
[12] E.O. Nielsen, U. Madsen, K. Schaumburg, L. Brehm, P. Krogsgaard-
Sample Preparation and Result Calculation
Larsen, Eur. J. Chem. – Chim. Ther. 1986, 21, 433–437.
A stock 1 mM solution of the test compound was prepared in 50% DMSO.
Subsequent dilutions were accomplished in buffer. The IC50 values were
calculated from 3–4 displacement curves based on 4–6 scalar concentrations
of the test compound in triplicate using the ALLFIT computer program[13]
and, in the case of tritiated glycine and AMPA binding, converted to Ki values
by application of the Cheng-Prusoff equation.[14]
[13] A. De Lean, P.J. Munson, D. Rodbard, Am. J. Physiol. 1978, 235,
E97–102.
[14] Y.-C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099–
3108.
Received: March 26, 1999 [FP380]
Arch. Pharm. Pharm. Med. Chem. 332, 201–207 (1999)