New non competitive AMPA antagonists

Article abstract of DOI:10.1016/S0968-0896(00)00133-4

New halogen atom substituted 2,3-benzodiazepine derivatives condensed with an azole ring on the seven membered part of the ring system of type 3 and 4 as well as 5 and 6 were synthesized. It was found that chloro-, dichloro- and bromo-substitutions in the benzene ring and additionally imidazole ring condensation on the diazepine ring can successfully substitute the methylenedioxy group in the well known molecules GYKI 52466 (1) and GYKI 53773 (2) and the 3-acetyl-4-methyl structural feature in 2, respectively, preserving the highly active AMPA antagonist characteristic of the original molecules. From the most active compounds (3b,i) 3b (GYKI 47261) was chosen for detailed investigations. 3b revealed an excellent, broad spectrum anticonvulsant activity against seizures evoked by electroshock and different chemoconvulsive agents indicating a possible antiepileptic efficacy. 3b was found to be highly active in a transient model of focal ischemia predictive of a therapeutic value in human stroke. 3b also reversed the dopamine depleting effect of MPTP and antagonized the oxotremorine induced tremor in mice indicating a potential antiparkinson activity. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00133-4

Bioorganic & Medicinal Chemistry 8 (2000) 2127±2143
New Non Competitive AMPA Antagonists
Gizella Abraham, Sandor Solyom,* Emese Csuzdi, Pal Berzsenyi, Istvan Ling,y
Istvan Tarnawa,{ Tamas Hamori, Istvan Pallagi, Katalin Horvath,x
Ferenc Andrasi, Gabor Kapus, Laszlo G. Harsing Jr., Istvan Kiraly,
Miklos Patthy and Gyula Horvath
Institute for Drug Research, Ltd, Berlini-u. 47-49, H-1045 Budapest, Hungaryk
Received 13 April 2000; accepted 24 May 2000
AbstractÐNew halogen atom substituted 2,3-benzodiazepine derivatives condensed with an azole ring on the seven membered part
of the ring system of type 3 and 4 as well as 5 and 6 were synthesized. It was found that chloro-, dichloro- and bromo-substitutions
in the benzene ring and additionally imidazole ring condensation on the diazepine ring can successfully substitute the methylene-
dioxy group in the well known molecules GYKI 52466 (1) and GYKI 53773 (2) and the 3-acetyl-4-methyl structural feature in 2,
respectively, preserving the highly active AMPA antagonist characteristic of the original molecules. From the most active com-
pounds (3b,i) 3b (GYKI 47261) was chosen for detailed investigations. 3b revealed an excellent, broad spectrum anticonvulsant
activity against seizures evoked by electroshock and di€erent chemoconvulsive agents indicating a possible antiepileptic ecacy. 3b
was found to be highly active in a transient model of focal ischemia predictive of a therapeutic value in human stroke. 3b also
reversed the dopamine depleting e€ect of MPTP and antagonized the oxotremorine induced tremor in mice indicating a potential
antiparkinson activity. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
The 2,3-benzodiazepine derivative GYKI 52466 (1) was
the prototype of the noncompetitive AMPA antagonists4
(Fig. 1). From our structure±activity relationship study
GYKI 53773 (2, LY300164, talampanel), a highly active
AMPA antagonist emerged from the 3-acylated 3,4-dihy-
dro analogues of 1,5 which is now one of the clinically
most advanced agents among the non competive AMPA
antagonists.6 Further structure±activity relationship stu-
dies revealed several structural features which are
important to maintain the potent AMPA antagonistic
character of the original molecules 1 and 2.7 We have
found that halogen atoms in the benzene ring can success-
fully substitute the dioxolane ring in 1 and 27d and the
biological activity was also retained when the 3-acyl-4-
methyl substitution pattern in 2 was replaced by some
nitrogen containing heterocycles attached to the 3,4-
positions of the 2,3-benzodiazepine ring system.7b In this
paper we report the synthesis and preliminary pharma-
cological studies of compounds 3±6, where halogen
atom(s) and condensed nitrogen containing ®ve membered
heterocycles were simultaneously applied to the 2,3-ben-
zodiazepine ring system.7e
Excessive activation of the glutamate receptors may be a
major factor in the pathologenesis of several acute and
chronic neurological disorders.1 Attention has been
focused to identify selective antagonists according to the
types of the ionotropic glutamate receptors classi®ed
according to their exogenous ligands N-methyl-d-aspartic
acid (NMDA), 2-amino-3-(3-hydroxy-methylisoxazol-4-
yl)propionic acid (AMPA) and kainic acid.1c Selective
AMPA antagonists have gained special importance
recently.2 Among them the noncompetitive antagonists
exerting their e€ect at an allosteric site of the receptor are
of particular interest because of the theoretical possibility
that they are e€ective even at extraordinarily high levels
of the natural transmitter glutamate, as well.2 Some of
the potential therapeutic targets of these antagonists
may be epilepsy, spasticity, pain, and neurodegenerative
disorders.3
*Corresponding author. Tel.:+36-1-399-3471; fax: +36-1-399-3356;
e-mail: h13759sol@ella.hu
yRecent address: EGIS, Kereszutuvi ut 30-38, Budapest, Hungary.
{Gedeon Richter Ltd, Gyomroi-u. 19-21, H-1103 Budapest, Hungary.
Chemistry Results
xPharmaceutical Control and Development Laboratory, Mexikoi u
 t 9,
H-1149 Budapest, Hungary.
One of the key steps in the synthesis of new 2,3-benzo-
diazepines with halogen atoms in the benzene ring is the
kA subsidiary of IVAX Corporation, Miami, Florida, USA.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00133-4

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
2128
Figure 1.
formation of isochromanes 9a±d from the correspond-
ing alcohols and an aldehyde (Scheme 1). While the
same reaction can be easily performed in the case of alco-
hols with electron donating groups, like, e.g. with 2-(3,4-
dimethoxyphenyl)- or 2-(3,4-methylenedioxyphenyl)eth-
anol or the corresponding isopropanols and 4-nitrobenz-
aldehyde using only slightly more than one equivalent of
concd hydrochloric acid,8 the analogous reactions with
7a±c need anhydrous conditions in benzene using
freshly molten zinc chloride and dry hydrogen chloride
gas and even then the yields are moderate. But on the
other hand the enhanced electrophilicity of the reacting
benzaldehyde derivative must contribute to the success
of the reaction as well, since, e.g. under the same reaction
conditions only negligible reaction could be noticed
between 7a and unsubstituted benzaldehyde. It was also
found e.g. with the less electrophilic 8b that apart from
the unknown steric contribution of the ortho substituent
to the reaction the corresponding isochromane 9d
formed with signi®cantly lower yield than the analogous
9b in the reaction between 7b and 8a.
have chosen the corresponding thiooxo derivatives 12a±d,
which were prepared from 11a±d by phosphorous penta-
sul®de in dry pyridine with acceptable yields.9 The latter
were reacted further with the known aminoacetals 13a±g.
This condensation step was most successful when the
reactants were heated in 2-methoxyethanol using red
mercury oxide as the sulfur binding agent. The resulting
intermediates of type 14 were generally puri®ed by column
chromatography and then further reacted with hydro-
chloric acid to give the imidazolo compounds 15a±g and
15i±n as well as 3p,q, respectively. The nitro groups in
benzodiazepines 15a±n were reduced by a standard method
using hydrazine hydrate and RaNi to give products 3a±n.10
Because of solubility considerations the reductions were
carried out generally in a mixture of methanol and di-
chloromethane. As imidazolo-benzodiazepines with
unsubstituted phenyl ring can hardly be synthesized by
the above route, because of the diculties concerning
the corresponding isochromane formation, this type of
compound was prepared by removal of the amino
group, e.g. in 3c by reaction with isoamyl nitrite and 5n
hydrochloric acid to provide 3r.11 In one instance the
aromatic amino group of 3b was acetylated to give 3o as
one of the potential metabolites. The prepared imidazolo
compounds are listed in Table 1.
The reaction of isochromanes 9a±d with Jones reagent in
acetone provided ketocarboxylic acids 10a±d. The latter
were reacted with excess hydrazine hydrate in ethanol to
give the intermediate hydrazones which were treated in
turn with dicyclohexylcarbodiimide to achieve ring clo-
sure. Another possibility to induce ring closing reaction
was the treatment of the intermediate hydrazone with
excess hydrochloric acid for longer time to provide the 4-
oxo-2,3-benzodiazepine derivative, e.g. 11a.
When 3b as one of the biologically most interesting
compounds had to be prepared in multigram scale we
became interested to avoid some disadvantages of the
synthetic route outlined in Scheme 1. These were the 5
step synthesis of the acetal 13b from alanine and the
optional but useful column chromatography of the
intermediates of type 14. It was attractive to make use
of a relatively scarcely used 4+1 cyclization method to
prepare the imidazole ring, where an acylamidoketone is
In order to enhance the reactivity of the 4-oxo compounds
11a±d towards condensation reactions to build up the
expected imidazolo- or triazolo-2,3-benzodiazepines, we

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
Â
2129
ꢀ
.
Scheme 1. Route I. (a) benzene, ZnCl2, HCl(g); (b) acetone, CrO3/H2SO4; (c) EtOH, H2NNH2 H2O, Á; (d) DCC or HCl; (e) pyridine, P2S5, 80ꢀ C;
ꢀ
.
(f) 2-methoxyethanol, red HgO, Á; (g) HCl, Á; (h) MeOH-CH2CL2, RaNi, H2NNH2 H2O; (i) DMF, isoamylnitrite, 65 C; (j) pyridine/AcCl, 5 C.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
2130
yield of this condensation step to the sensitive nature of
the acetone side chain in 16 towards reactions, since
with the phenacyl derivative 17 the same condensation
could be performed with a signi®cantly better yield
(82%). (Scheme 2.)
For the formation of the triazolo ring in compounds 4a±d
acylhydrazides were used (Scheme 3).13 Better results
could be achieved in the condensation steps when instead
of thione 12a the corresponding 4-methylthio-2,3-ben-
zodiazepine 18 was used and additionally a catalytic
amount of hydrochloric acid was applied, as well. The
nitro groups in compounds 19a±d were reduced by
the standard RaNi-hydrazine hydrate method and the
resulting compounds 4a±d are shown in Table 2.
To build up the 11H-imidazolo[3,4-c][2,3]benzodiazepine
ring system in 5 we started from 23 which was synthesized
on the analogy to well established methods (Scheme 4).14
The methyl group in 23 was oxidized by selenium diox-
ide to form the corresponding aldehyde 24, which was
then reduced to the alcohol 25 by sodium borohydride. The
Scheme 2. Route II. (a) DMF, K2CO3; (b) NH4OAc, AcOH, Á.
reacted with ammonium acetate as nitrogen atom donor
in acetic acid.12 The necessary amidoketone 16 was pre-
pared in very good yield by alkylation of 11a with chlor-
oacetone (Scheme 2). We have found that condensation
of 16 to give the corresponding imidazolo-benzodiaze-
pine 15b needs a large excess (>50 equivalents) of
ammonium acetate in acetic acid and the yield is modest
(40%). The use of other ammonium salts like formate or
carbonate as well as solvents, e.g. formamide, propionic
acid, tri¯uoroacetic acid or microwave techniques
instead of conventional heating did not give signi®cant
improvement. Despite the lower yield of the imidazole
forming step, 3b can be produced by this route without
any laborious synthetic steps. We attribute the lower
Table 1. 11H-Imidazolo[1,2-c][2,3]benzodiazepine derivatives (3a±r)
Compound X Y R1
R2
R3
R4
mp
(ꢀC)
Yield
(%)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H Cl
H Cl Me
H Cl
H Cl Me
H Cl Et
H
H
H
Me
Me
H
H
H
H
H
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
210±214 67
229±230 79
267±270 71
274±278 83
247±250 72
250±253 84
293±294b 68
223±226 81
254±255 40
284±286 71
248±251 64
263±268 77
272±275 84
295±300b 41
H
H Cl
H Cl
H
H
4-NH2-C6H4Ð H
4-pyridyl
H
H
H
H
H
H
H
H
Scheme 3. (a) acetone, MeJ, K2CO3; (b) DMF, acylhydrazine, cat.
.
HCl; (c) MeOH-CH2Cl2, RaNi, H2NNH2 H2O.
H Cl Ph
Cl Cl Me
H
H
Me
H
Me
Me
4-pyridyl
H
Cl Cl
H Br Me
H Br
H Br Me
H Br
H
H
Table 2. 6-(4-Aminophenyl)-8-chloro-11H-1,2,4-triazolo[4,5-c][2,3]
benzodiazepine derivatives (4a±d)
H
Compound
R
mp (ꢀC)
Yield (%)a
H Cl Me
H Br Me
H NHCOMe 265±266 63
H
Me
Me
Cl
Cl
H
H
H
H
132±140c 21
210±215c 48
166±169 39
H Br
H Cl
H
H
4a
4b
4c
4d
Me
4-pyridyl
4-NH2-C6H4Ð
CH3OCH2Ð
228±231
284±288
191±193
195±197
91
92
85
83
aYields refer to the last step of the synthesis.
bDecomposition.
cHydrochloride salts.
aYields refer to the last step of the synthesis.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
Â
2131
.
Scheme 4. (a) 4-nitrobenzaldehyde, benzene, ZnCl2, HCl(g); (b) acetone, CrO3/H2SO4, then 70% HClO4; (c) DMF, H2NNH2 H2O; (d) dioxane,
.
SeO2; (e) THF-H2O, NaBH4; (f) THF, Ph3P, phthalimid, DEAD; (g) MeOH, H2NNH2 H2O; (h) Ac2O; (i) ClCH2CH2Cl, POCl3, Á; (j) MeOH-
.
CH2Cl2, RaNi, H2NNH2 H2O.
latter was transformed under Mitsunobu conditions15 with
phthalimide into 26 then hydrazinolysis and acetylation
gave 27 and 28, respectively. Cyclization of 28 with phos-
phorus oxychloride gave 29 which was reduced by RaNi-
hydrazine hydrate to give 5.
The pyrrolo derivative 6 was prepared from 23 by using a
combined alkylation-condensation procedure16 with ethyl
bromopyruvate to give the nitro compound 30 which on
reduction provided 6 (Scheme 5).
Scheme 5. (a) Br-CH2-C(O)-CO2Et, EtOH, Á; (b) MeOH-CH2Cl2,
.
RaNi, H2NNH2 H2O.
Biological Results and Discussion
Primary pharmacological testing and structure±activity
relationships
Inhibition of AMPA-, or kainate-triggered spreading de-
pression in isolated chicken retina. Excitation of isolated
chicken retinas by glutamate receptor agonists provokes
spreading depression (SD), which is accompanied by a
characteristic change of the light scattering properties of
the preparation. The phenomenon is easily visible by
unaided eye, thus it is a convenient method for drug
Table 3 contains primary in vitro and in vivo biological
data with the newly synthesized compounds in comparison
with the standard molecules 1 (GYKI 52466) and 2
(GYKI 53773, LY300164, talampanel).

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
2132
Table 3. Screening results of the condensed 2,3-benzodiazepine derivatives 3±6a
Compond
Behavioural changes
(100 mg/kg ip; 200 mg/kg po)
Retinal spreading depr.
IC50 (mM)
MES Inc. screen
ED50, mg/kg po ED50, mg/kg ip
1
2
Loss of righting re¯ex
Loss of righting re¯ex
Short loss of righting re¯ex
Loss of righting re¯ex
Ataxia, muscle relaxation, po: loss of righting re¯ex
Ataxia
A: 6.3 K: 9.5
A: 1.7 K: 2.6
A: 6.5 K: 3.8
37.4
8.6
61.6
24.0
47.1
13.4
47.1
36.5
100-125
>200
>150
ꢁ200
>200
>200
47.2
3a
3b
3c
3d
3e
3f
3g
3h
3i
A: 7.3 K: 2.5
A: 4.1 K: 0.5 (¯at curve)
A: 11.8 K: 1.5
61.3
ꢁ100
Loss of righting re¯ex, ataxia, SMA#
A: >20 K: >20
A: 4.3 K: 7.1
A: 3.1 K: n.t.
A: >20 K: n.t.
A: 3.1 K: 1.9
44.6
SMA#, ataxia
>100
>100
>100
24.0
50±100
40.3
ꢁ100
ꢁ100
>100
56.0
>100
>100
>100
ꢁ100
>100
>100
>100
ꢁ100
>100
SMA#, ataxia
1
Loss of righting re¯ex
po: SMA#, ataxia
Loss of righting re¯ex
SMA#, ataxia, weak muscle relaxation
SMA#, ataxia
3j
3k
3l
A: >20 (0.6±10 mM: max. 47% inhibition) K: 1.1
A: 9.3 K: 2.2
A: 4.0 K: 1.3
>200
68.4
ꢁ200
>200
>200
>200
>200
ꢁ200
>200
ꢁ100
>200
>200
ꢁ150
>200
ꢁ200
3m
3n
3o
3p
3q
3r
4a
4b
4c
4d
5
A: >20 K: 7.4
A: 3.2 K: >20
po: SMA#
SMA#, ataxia
A: >20 K: >20
A: >20 K: >20
A: >20 K: >20
A: >20 K: >20
A: 2.9 K: 4.1
ip: Weak SMA#
ip: SMA#
ip: Weak SMA#
Ataxia, weak muscle relaxation
1
A: >20 K: ꢁ20
1
A: >20 K: 1.0
A: 9.2 K: 5±10
SMA#, ataxia
ip: SMA#, ataxia
A: >20 K: n.t.
A: >20 K: n.t.
6
100 ip.: 1
aSMA#: decrease of spontaneous motor activity; A: AMPA; K: kainate; n.t.: not tested.
testing. Retinal SD can be blocked by glutamate
antagonists.17 In our study the majority of the tested
compounds were e€ective in the retinal spreading
depression (SD) test, i.e. blocked either AMPA- or kai-
nate induced SD with an IC50 lower than 10 mM. The
IC50 values of the most potent compounds were similar
to those of 1 or 2 in the AMPA test, and lower in the
kainate test. Compounds 3b,d,j,k,l,m seemed to show
some selectivity towards kainate responses, i.e. their IC50
values were more than 2-fold higher against AMPA
than against kainate. The concentration-response curves
of 3b against the two agonists are shown in Fig 2.
Interestingly, some compounds such as 3e, 3m, or 4d
caused only partial inhibition of kainate-triggered SD
(maximal inhibition: 40±60% at 20mM; not shown), or had
a ¯at dose-response curve (3a, 3c, or 3i; not illustrated).
The signi®cance of the di€erences between AMPA and
kainate antagonistic actions in the case of some com-
pounds is dicult to tell, as the role of the speci®c kainate
receptors in triggering SD in this preparation has not been
characterized yet. It is to be noted here that the competitive
AMPA antagonist 1,2,3,4-terahydro-6-nitro-2,3-dioxo-
benzo[f]quinoxaline-7-sulfonamide (NBQX) also had a
¯at dose-response curve against kainate in this model.17
Regarding the dose-response curves in the AMPA-
induced spreading depression test, the curves were usually
steeper than in the kainate test, except for 3j which only
partially depressed AMPA response (maximum inhibition:
45% at 2.5 mM).
Behavioral changes, e€ects of the compounds in the max-
imal electroshock (MES) test, and in the inclined screen
test. Most compounds elicited some gross behavioral
e€ects after ip (100 mg/kg) or po (200 mg/kg) applica-
tion, indicating a reasonable absorption and penetration
through the blood brain barrier. These included a
decrease in motility, ataxia, muscle weakness and even a
loss of righting re¯ex with the most e€ective substances.
These behavioral symptoms have also been observed
with other 2,3-benzodiazepine compounds5a and can be
regarded as the consequence of the blockade of central
AMPA receptors function.
AMPA receptor blockade also results in an antic-
onvulsant action.2,5a MES test has been found a suitable
method for establishing in vivo structure±activity rela-
tionships among another group of 2,3-benzodiazepine
Fig. 2. Inhibition of kainate- and AMPA induced spreading depres-
sion in isolated chick retina by 3b. & 5 mm AMPA; ^ 5 mm kainate.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
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2133
AMPA antagonists.5a The two most e€ective compounds
of the present series were 3b and 3i, both with an ED50
of 24 mg/kg po, which is between the ED50 values of 1
and 2.
pretreatment was applied. Similar metabolic activation
was described among other 2,3-benzodiazepine AMPA
antagonists.5a
Similarly to earlier 2,3-benzodiazepine type AMPA
antagonists5a,7 the ecacy of the present compounds is
also generally restricted to the 4-aminophenyl derivatives
(see e.g. the dezamino compounds 3p,q,r), and even an
acetylation of the amino group (e.g. in 3o) decreases
biological activity.
The ataxia inducing or muscle relaxant action was
quanti®ed in the inclined screen assay. The ED50 values
of the compounds in this test (30 min ip pretreatment)
were in general slightly higher than in the MES test (60
min oral pretreatment). A bigger gap between the
anticonvulsant and motor impairment inducing doses
would be expected if identical treatment schedules were
used. This MES preference does not hold true in respect
of 3a, which was more potent in the muscle relaxant
assay. A possible explanation for this discrepancy is that
the compound seems to have an extremely short duration
of action (probably due to a rapid metabolism in mice),
and 60 min after the treatment its blood concentration
decreases to a level that can not protect the animal from
MES. Another possible explanation is thatÐin contrast
to most of the 2,3-benzodiazepines3Ð3a has a poor
absorption from the intestines.
On the basis of the screening results 3b and in certain
cases additionally 3i were chosen for more detailed and
comparative investigations.
Inhibition of AMPA-, or kainate-induced whole-cell cur-
rents by 3b in isolated neurons. The AMPA receptor
antagonistic e€ect of 3b in freshly isolated cerebellar Pur-
kinje cells is shown in Fig. 3. In this preparation 3b is
nearly equiactive with 2 and more potent than 1. Its IC50
against AMPA (2.3 mM) was somewhat lower than
against kainate (4.5mM, not shown). However, under our
experimental conditions most current elicited by kainate
was probably mediated by AMPA receptors. Thus these
data are not suitable for analyzing a possible subtype
selectivity of the compound. The current blocking
action of 3b (10 mM) against 5 mM AMPA developed
slowly and the recovery from the blockade took several
seconds (ton and to€ of the current trajectories were
4.2Æ0.8 and 5.2Æ0.7 sec, respectively), suggesting con-
siderably slower binding and unbinding kinetics than
those of 1 and the racemate of 2 (ton and to€ values
below 1 sec).18 To assess the biological signi®cance of
this feature of 3b warrants further studies. Just as 1 or
2,18,19 3b acted in a non-competitive way, as its current
blocking action was not dependent on the agonist con-
centration (not shown).
Correlating the in vitro and in vivo pharmacological
data with the structural features of the new compounds
the following relationships can be established. Applying
chloro-, dichloro- or bromo-substitution instead of the
methylendioxy group of molecules 1 and 2 and substituting
simultaneously the hydrogen bond acceptor type acetyl
group of 2 by a condensed nitrogen containing ®ve mem-
bered heterocycle preserve the high AMPA antagonist
character of the original molecules. Even among the
bromo compounds we found a compound (3k) with sig-
ni®cant activities.
Concerning the condensed heterocycles optimum ecacy
was found among the imidazolo[1,2-c][2,3]benzodiaze-
pines of type 3. Although most of the triazolo-derivatives
4a±d showed acceptable in vitro ecacy, the additional
hydrogen bond acceptor atom in the condensed ring may
have prevented a proper absorption resulting in an in
vivo inactivity. With pyrrolo-derivative 6, where no
hydrogen bond possibility exists in a similar way as it
may occur for molecules 3, 4 and 2 or where the acceptor
nitrogen atom is probably at a wrong place (e.g. imida-
zolo[3,4-c][3,4]benzodiazepine derivative 5) neither in
vitro nor in vivo e€ects were found.
Anticonvulsant pro®le of selected 2,3-benzodiazepines.
The broad-spectrum anticonvulsant activity of 1 and 2
has already been described.3,5a Table 4 illustrates that
the anticonvulsant pro®le of 3b and 3i is similar to the
former compounds. Their potencies were usually
between those of 1 and 2. It is notable, however, that
both newly synthesized compounds protected mice from
Within the imidazolo-derivatives 3 the optimum ecacy
was found among the compounds substituted with little
aliphatic groups (3b,c,e,i,k). Bigger aryl substitutions
resulted in inactive or nearly inactive compounds, e.g.
3f,g,h,n, whereas methyl substitution in position 3 seems
to enhance kainate selectivity, e.g. 3c,d,j,l. But double
methyl substitution, e.g. in 3d and 3m, resulted in loss of
in vivo activity, which may be explained by a worsened
ADME pro®le of these derivatives. It is worth mentioning
that 3e, a homologue of one of the most active sub-
stances (3b), is inactive in vitro but still has a reasonable
anticonvulsant activity. Formation of biologically active
metabolites may underlie this discrepancy, which is
supported by the observation that the compound was
ine€ective in the inclined screen assay, where a shorter ip
Fig. 3. Inhibition of AMPA induced whole cell current in freshly iso-
lated cerebellar Purkinje cells by 3b.

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2134
Table 4. Anticonvulsive e€ects of selected compounds (3b and 3i) against MES and various chemical convulsants
ED50 mg/kg po
1
2
3b
3i
MES
Metrazole
Strychnine
Bemegride
Bicuculline
Nicotine
4-aminopyridine
3-mercapto-propionic acid
37.4 (29.2±47.5)
119.8 (108.5±132.3)
86.7 (71.7±104.9)
71.9 (62.0±83.4)
35.0 (28.5±43.1)
71.8 (49.8±103.7)
43.0 (39.9±59.8)
47.0 (32.9±67.2)
8.6 (7.0±10.6)
16.8 (10.2±27.6)
17.4 (10.6±28.4)
23.9 (16.0±35.7)
14.6 (5.8±36.7)
22.7 (16.2±31.9)
8.4 (5.6±12.5)
24.0 (17.9±32.1)
53.8 (43.7±66.2)
67.4 (43.7±66.2)
40.7(21.9±56.9)
22.8 (16.7±31.2)
13.1 (8.1±21.2)
55.5 (47.1±65.4)
38.1 (28.7±50.5)
24.0 (17.9±32.1)
62.2 (44.1±87.6)
44.2 (38.5±50.7)
39.9 (26.5±60.1)
22.5 (13.4±37.7)
18.3 (14.2±23.8)
6.7 (3.5±12.7)
17.1 (9.7±30.1)
66.8 (43.0±103.1)
nicotine-induced seizures and death more potently than
2 did, which may indicate a potential antiparkinsonian
ecacy. Further, 3i was the most e€ective of the four
compounds in preventing 4-aminopiridine (4-AP)-induced
seizures.
Table 6. E€ect of 3b on the size of ischemic damage in rats after
transient occlusion of the medial cerebral artery
Comp.
Doses
(mg/kg iv)
Infarct size scores
(mean Æ S.E.)
Decrease
(%)
Veh.a
1
48920Æ4012
32218Æ6325*
40518Æ5928
13229Æ2313*
42795Æ7513
16122Æ4368*
6Â5
6Â2
6Â1
34.1
67.4
62.3
Muscle relaxant e€ect of selected 2,3-benzodiazepines. A
common feature among 2,3-benzodiazepine AMPA
antagonists is the inhibition of motor functions with a
primarily spinal site of action.3,20 The ataxia/muscle
relaxation inducing ED50 values of 3b and 3i are listed
and compared to those of 1 and 2 in the inclined screen
and rotarod tests (Table 5).
Veh.a
3b
Veh.a
3b
aVehicle. * P<0.01 Signi®cance was calculated by one-way ANOVA
followed by the Duncan test. Treatment schedule: ®rst injection 30
min after occlusion and repeated 5 times in every 30 min.
Antiischemic e€ect of 3b in a transient focal ischemia
model, in rats. The antiischemic property of AMPA
antagonists renders them promising drug candidates in
various acute and chronic neurodegenerative disorders
such as stroke.1 3 Compound 1 has been shown to pro-
tect animals from ischemic damage in various models.21
The transient focal ischemia model is a relevant stroke
model.22,23 Table 6 shows that 3b, applied at 2.5±5-fold
lower doses, had a considerably more potent e€ect on
infarct size than 1.
Table 7. Antagonism of oxotremorine induced tremor in mice
Oxotremorine antagonism ED50 mg/kg po
1
2
3b
20.5 (14.9±28.3)
5.6 (3.6±8.5)
16.8 (12.0±23.6)
.
N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine HCl
(MPTP) neurotoxicity. Degeneration of dopaminergic
neurons in Parkinsons disease is well known.24 Recent
®ndings indicate that impairment of striatal dopami-
nergic neurotransmission results in an overactivity of
glutamatergic neurons in the subthalamic nucleus.25
This glutamatergic overactivity is thought to play an
important role in the expression of some parkinsonian
symptoms, such as hypokinesia and rigidity as well as in
development of neuronal damage.26 Therefore we tested
the e€ects of 3b against MPTP-induced neurotoxicity in
mice, a relevant rodent model of Parkinsons disease, in
comparison with those of 1 and of the competitive
AMPA antagonist NBQX.
The antiparkinsonian e€ects of 3b
Oxotremorine induced tremor. The cholinergic agonist
oxotremorine, applied systemically, provokes tremor
with a central action. Table 7 shows that the 2,3-benzo-
diazepine compounds 1, 2 and 3b potently mitigated the
tremor induced by oxotremorine. Salivation, a peripheral
e€ect of oxotremorine, was not a€ected by these drugs.
Since Parkinson's disease is characterized by a disturbed
balance between cholinergic and dopaminergic neuro-
transmission in the basal ganglia, an indirect central
anticholinergic e€ect of compounds 1, 2 and 3b may be
indicative for a therapeutically suitable antiparkinsonian
e€ect.
Table 5. Muscle relaxant e€ects of selected compounds (3b and 3i) in the inclined screen and rotarod tests, in mice
ED50 mg/kg ip
Method
1
2
3b
3i
Inclined screen
Rotarod
47.1 (44.2±50.2)
24.0 (22.0±26.2)
13.4 (11.2±16.0)
2.3 (1.6±3.4)
36.5 (29.4±45.2)
15.8 (13.0±19.3)
47.2 (41.9±53.3)
13.7 (12.4±15.1)

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
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2135
Table 8. E€ects of 3b on MPTP induced depletion of dopamine and its metabolites in mouse striatuma
Treatment
Concentration (mg/g) of
1
0 min.
2
30 min.
3
150 min.
4
270 min.
5
390 min.
DA
DOPAC
HVA
Vehicle
MPTP
MPTP
MPTP
MPTP
Vehicle
Vehicle
3b
1
NBQX
Vehicle
Vehicle
3b
1
NBQX
Vehicle
Vehicle
Vehicle
1
Vehicle
Vehicle
3b
Vehicle
Vehicle
11.14Æ0.43
1.07Æ0.17b
4.38Æ0.89c
3.60Æ0.37c
2.74Æ0.52d
0.54Æ0.03
0.10Æ0.04b
0.21Æ0.05
0.27Æ0.03d
0.16Æ0.04
0.87Æ0.05
0.33Æ0.06b
0.45Æ0.06
0.36Æ0.03
0.37Æ0.03
NBQX
aSigni®cance was calculated by one-way ANOVA followed by the Duncan's test.
b<0.01 versus saline treated control group.
cP<0.01 versus MPTP treated group.
dP<0.05 versus MPTP treated group.
Table 9. Determination of acute toxicity in mice
spectrum anticonvulsant activity against seizures evoked
by electroshock and di€erent chemoconvulsive agents
indicating a possible antiepileptic ecacy. 3b (GYKI
47261) was also found to be highly active in a transient
model of focal ischemia predictive of a therapeutic value
in human stroke. A signi®cant e€ect antagonizing the
MCAO induced infarct size in rat brain was found in a
®ve times lower dose than for GYKI 52466 (1), the repre-
sentative of the non competitive AMPA antagonists. In
addition 3b reversed the dopamine depleting e€ect of
MPTP and antagonized the oxotremorine induced tremor
in mice. These ®ndings indicate that the non-competitive
AMPA antagonist 3b may have therapeutical potential
not only in acute but chronic neurodegenerative disorders
as well.27
LD50 mg/kg
Compound
ip
po
1
3b
302.1 (273.9±333.3)
259.4 (205.8±327.1)
296.9 (258.6±340.8)
372.9 (322.5±431.1)
The e€ects of 3Â20 mg/kg (ip dose) of 3b, 1 and NBQX
on MPTP-induced dopamine (DA) depletion in mouse
striatum are shown in Table 8. MPTP injection reduced
DA concentration in the striatum by 90% and the levels of
DA metabolites, dihydroxyphenylacetic acid (DOPAC)
and homovanillic acid, (HVA) were also decreased sig-
ni®cantly (Table 8). MPTP administration did not a€ect
the concentrations of serotonin and 5-hydroxyindoleacetic
acid (5-HIAA) in the striatum indicating the speci®c toxic
e€ect of MPTP on dopaminergic neurons (data not
shown). 3b and 1, when they were injected repeatedly,
reversed the MPTP-induced decrease in striatal con-
centrations and NBQX exhibited similar e€ect (Table 8).
The neuroprotective e€ects of 2,3-benzodiazepines against
MPTP neurotoxicity suggest that non-competitive
AMPA receptor antagonists may have a bene®cial role
in the treatment of Parkinson's disease.25,26
Experimental
Chemistry
Melting points were measured on a Boetius hotstage
microscope and are uncorrected. IR (KBr): Bruker IFS-85
1
spectrophotometer. HNMR (CDCl3, internal standard
TMS,T=298ꢀK): Bruker AC 250, other solvents are
indicated. Mass spectra: Finnigan MAT 8430 mass
spectrometer. Operating conditions: electron ionization,
Eel=70eV, Iel=0.5 mA, Uacc=3 kV, R=1250. FAB:
ION-TECH atom gun with Xe, in m-nitrobenzyl alcohol
matrix. Column chromatography: silica gel, Kieselgel 60,
Merck. Satisfactory elemental analyses (Æ0.4 %) for C, H,
N and S were obtained for all new described compounds
except for 25±29 which were not measured this way. The
following compounds were prepared according to the lit-
erature: 7a,28 7b,28 7c,29 13a,30 13b,31 13c,30 13d,30 13e,31
13f,32 13g.32
Acute toxicity. Compound 3b caused lethality in mice
only at doses several fold higher than its ED50 values in
the pharmacological assays. Its toxicity is similar to that
of 1. The LD50 values of 3b and 1 were almost the same
after ip and oral administrations suggesting a good
gastro-intestinal absorption (Table 9).
Conclusion
Among the newly synthesized, with heterocycle condensed
and halogen substituted 2,3-benzodiazepine derivatives
several non-competitive AMPA antagonists were identi-
®ed with potencies that approached or exceeded those of
the reference compounds 1 or 2. Some of them also had
potent in vivo activity. 3b, the compound selected for
detailed studies, displayed a pharmacological pro®le
largely similar to that of the formerly described 2,3-
benzodiazepine AMPA antagonists,3 but a potent anti-
parkinsonian activity was also revealed as an additional
feature. The compound showed an excellent, broad
General procedure for the synthesis of isochromanes 9a±d.
To a stirred solution of the phenylethanol derivative
(7a±c) (0.1 mol) and the corresponding benzaldehyde
8a,b (0.1 mol) in dry benzene (300 mL) freshly molten
and ground zinc chloride (13.6 g, 0.1 mol) was given.
Dry hydrogen chloride gas was introduced into the sus-
pension over 4 h and the mixture was stirred overnight.
The resulting mixture was then washed with water and a
5% sodium-hydrogensul®t solution. The organic phase
was dried and after ®ltration the solvent evaporated.
The residue was recrystallized to give the title products.

Â
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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
2136
7-Chloro-1-(4-nitrophenyl)isochromane (9a). Yield: 56%.
Mp 98±101ꢀC (ethanol). C15H12ClNO3 (289.7). 1H
NMR: d 2.79 (ddd, J1=16.5 Hz, J2=J3=3.6 Hz, 4-H)
and 3.12 (ddd, J1=16.5 Hz, J2=9.7 Hz, J3=5.6 Hz, 4-H),
3.92 (ddd, J1=11.5 Hz, J2=9.7 Hz, J3=3.6Hz, 3-H) and
4.21 (ddd, J1=16.5 Hz, J2=5.6 Hz, J3=3.6Hz, 3-H), 5.75
(s, 1H, 1-H), 6.65 (d, J=1.9Hz, 1H, 8-H), 7.13 (d,
J=8.3Hz, 1H, 5-H), 7.17 (dd, J1=8.3 Hz, J2=1.9Hz,
1H, 6-H), 7.50 (d, J=8.8 Hz, 2H) and 8.25 (d, J=8.8Hz,
2H, nitrophenyl).
J2=2.0 Hz, 1H, 5-H), 7.95 (d, J=8.9 Hz, 2H) and 8.38
(d, J=8.9 Hz, 2H, nitrobenzoyl), 12.1±12.7 (1H).
4,5-Dichloro-2-(4-nitrobenzoyl)phenylacetic acid (10c).
The crude product prepared by the evaporation of its
solution in acetone was recrystallized from 96% acetic
acid and further puri®ed by column chromatography.
Eluent: chloroform:methanol (9:1). Yield: 54 %. Mp 187±
190 ꢀC. C15H9Cl2NO5 (354.1). H NMR: (DMSO-d6): d
1
3.90 (s, 2H, CH2), 7.68 (s, 1H), 7.85 (s, 1H), 7.97 (d,
J=8.7Hz, 2H) and 8.38 (d, J=8.7 Hz, 2H, nitrobenzoyl).
7-Bromo-1-(4-nitrophenyl)isochromane (9b). Yield: 62%.
Mp 104±107 ꢀC (ethyl acetate). C15H12BrNO3 (334.2). 1H
NMR: d 2.78 (ddd, J1=16.2 Hz, J2=J3=3.6 Hz, 4-H)
and 3.12 (ddd, J1=16.2 Hz, J2=9.9 Hz, J3=5.8 Hz, 4-H),
3.93 (ddd, J1=13.5 Hz, J2=9.9 Hz, J3=3.6Hz, 3-H) and
4.21 (ddd, J1=13.5 Hz, J2=5.8 Hz, J3=3.6Hz, 3-H), 5.78
(s, 1-H), 6.82 (d, J=1.9 Hz, 8-H), 7.10 (d, J=8.2 Hz, 5-H),
7.35 (dd, J1=8.2Hz, J2=1.9Hz, 6-H), 7.55 (d, J=8.8Hz,
2H) and 8.22 (d, J=8.8 Hz, 2H, nitrophenyl).
4-Bromo-2-(2-chlorobenzoyl)phenylacetic acid (10d).
Yield: 47%. Mp 135±138 ꢀC. C15H10BrClO3 (353.6). H
NMR: (DMSO-d6): 3.90 (s, 2H, CH2), 7.37 (d, J=2.2Hz,
1H, 3-H), 7.43 (d, J=8.1Hz, 1H, 6-H), 7.78 (dd,
J1=8.1 Hz, J2=2.2 Hz, 1H, 5-H), 7.47 (m, 2H) and 7.60
(m, 2H, chlorophenyl), 12.40 (br. s).
1
8-Chloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-
epin-4-one (11a). Method A. A solution of 10a (17.6 g,
55.0 mmol) and 85% hydrazine hydrate (8 mL) in etha-
nol (340 mL) was heated at re¯ux for 4 h. The mixture
was then chilled, treated with 1N hydrochloric acid
(115 mL) and the solvent was evaporated. The residue was
treated with water (50 mL) and the precipitate ®ltered and
dried. This intermediate hydrazone was dissolved in di-
chloromethane (300mL) and treated with a solution of
dicyclohexylcarbodiimide (13.4 g 65.0 mmol) in dichloro-
methane (210mL). The mixture was stirred overnight at rt
and the resulting precipitate was ®ltered and washed
with dichloromethane to give 11a (12.5 g). Yield: 72 %.
Mp 275±278 ꢀC.
7,8-Dichloro-1-(4-nitrophenyl)isochromane (9c). Yield:
30%. Mp 130±132ꢀC (ethanol). C15H11Cl2NO3 (324.1). 1H
NMR: d 2.80 (ddd, J1=16.9 Hz, J2=J3=3.9 Hz, 4-H) and
3.13 (ddd, J1=16.9 Hz, J2=10.0 Hz, J3=5.9 Hz, 4-H),
3.91 (ddd, J1=11.5 Hz, J2=10.0 Hz, J3=3.9 Hz, 3-H) and
4.20 (ddd, J1=11.5 Hz, J2=5.9 Hz, J3=3.9Hz, 3-H), 5.72
(s, 1H, 1-H), 6.76 (s, 1H, 8-H), 7.30 (s, 1H, 5-H), 7.48 (d,
J=8.7 Hz, 1H) and 8.23 (d, J=8.7 Hz, 1H, nitrophenyl).
7-Bromo-1-(2-chlorophenyl)isochromane (9d). Yield:
40%. Mp 62±65 ꢀC (ethanol). C15H12BrClO (323.6). H
1
NMR: d 2.75 (ddd, J1=16.4 Hz, J2=J3=3.6 Hz, 4-H)
and 3.09 (ddd, J1=16.4 Hz, J2=9.7 Hz, J3=5.7 Hz, 4-H),
3.93 (ddd, J1=13.6 Hz, J2=9.7 Hz, J3=3.6Hz, 3-H) and
4.21 (ddd, J1=13.6 Hz, J2=5.7 Hz, J3=3.6Hz, 3-H), 6.88
(d, J=1.7Hz, 1H, 8-H), 7.05 (d, J=8.0 Hz, 1H, 5-H),
7.14±7.34 (m, 4H, 2-clorophenyl), 7.45 (dd, J1=8.0Hz,
J2=1.7Hz, 1H, 5-H).
Method B. The reaction was performed as in Method A,
but isopropanol was used as solvent and after re¯ux it was
treated with 1N hydrochloric acid (125mL) and stirred
overnight at rt The resulting precipitate was ®ltered and
the crude product was recrystallized from 2-methox-
yethanol. Yield: 88%. Mp 275±277 ꢀC. C15H10ClN3O3
1
General procedure for the synthesis of phenylacetic acid
derivatives 10a±d. To a solution of the corresponding
isochromane (9a±d) (90 mmol) in acetone (360 mL)
Jones reagent (260 mL) was gradually given and the
mixture was stirred for 16 h. The separated chromium
sulfate was ®ltered and the ®ltrate evaporated to dry-
ness. The residue was treated with excess 10% sodium
carbonate solution and dichloromethane. After separa-
tion the aqueous layer was acidi®ed with concd hydro-
chloric acid and the separated crystals were collected by
®ltration.
(315.7). H NMR: (DMSO-d6): d 3.62 (s, 2H, 5-H), 7.20
(d, J=2.0 Hz, 1H, 9-H), 7.58 (d, J=8.2Hz, 1H, 6-H), 7.70
(dd, J1=8.2Hz, J2=2.0Hz, 1H, 7-H), 7.80 (d, J=8.7Hz,
2H) and 8.30 (d, J=8.7 Hz, 2H, nitrophenyl).
8-Bromo-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-
epin-4-one (11b). Starting from 10b (12.7 g, 35.0 mmol)
Method A was essentially followed as described for 11a.
Yield: 65% (8.19 g). Mp 264±267 ꢀC. C15H10BrN3O3
1
(360.2). H NMR: (DMSO-d6): d 3.60 (br. s, 2H, 5-H),
7.29 (d, J=1.8 Hz, 1H, 9-H), 7.52 (d, J=8.3 Hz, 1H, 6-
H), 7.77 (overlapping, 7-H), 7.78 (d, J=8.7 Hz) and 8.32
(d, J=8.7 Hz, 2H, nitrophenyl), 11.41 (s, 1H, N-H).
4-Chloro-2-(4-nitrobenzoyl)phenylacetic acid (10a). Yield:
78%. Mp 150±152 ꢀC (ethyl acetate). C15H10ClNO5
(319.7). 1H NMR: (DMSO-d6): d 3.82 (s, 2H, CH2), 7.42
(d, J=1.8 Hz, 1H, 3-H), 7.48 (d, J=8.2Hz, 1H, 6-H), 7.62
(dd, J1=8.2Hz, J2=1.8 Hz, 1H, 5-H), 7.90 (d, J=8.7Hz,
2H) and 8.32 (d, J=8.7 Hz, 2H, nitrobenzoyl).
7,8-Dichloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzo-
diazepin-4-one (11c). A solution of 10c (6.1 g, 17.2 mmol)
and 85% hydrazine hydrate (6 mL) in isopropanol
(300 mL) was treated at re¯ux for 6 h. The solvent was
evaporated and the residue was dissolved in a mixture of
40% acetic acid (40 mL) and dichloromethane (400 mL).
After separation the organic layer was washed with
water, dried and treated with dicyclohexylcarbodiimide
(3.60 g 17.5 mmol). The reaction mixture was stirred
4-Bromo-2-(4-nitrobenzoyl)phenylacetic acid (10b). Yield:
62 %. Mp 128±130 ꢀC. C15H10BrNO5 (364.2). 1H NMR:
(DMSO-d6): d 3.80 (s, 2H, CH2), 7.46 (d, J=8.2 Hz, 1H,
6-H), 7.57 (d, J=2.0 Hz, 1H, 3-H), 7.81 (dd, J1=8.2 Hz,

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
Â
2137
overnight and the precipitate ®ltered. The ®ltrate was
evaporated to dryness and the residue was boiled with
methanol (120mL) and ®ltered while hot to give the pro-
duct (4.40 g). Yield: 73%. Mp 268±270 ꢀC. C15H9Cl2N3O3
(350.1). 1H NMR: (DMSO-d6): d 3.76 (s, 2H, 5-H), 7.48 (s,
1H, 6-H), 7.90 (d, J=8.8 Hz, 2H) and 8.39 (d, J=8.8 Hz,
2H, nitrophenyl), 8.01 (s, 1H, 9-H).
removed and the residue was puri®ed by chromatography,
eluent was chloroform:methanol (98:2). The fractions
containing the condensation product of type 14 were eva-
porated to dryness and the residue was treated with a 1:1
mixture of concd hydrochloric acid and ethanol and
heated at re¯ux for 1±2 h. Evaporation gave the title pro-
ducts as hydrochloride salts. In a few cases (15f,g,n) the
intermediate condensation product was reacted with
methanesulfonic acid at rt for 1±2 h to induce the ring
closure reaction. In these cases the reaction mixture was
diluted with water and made alkaline with 5N sodium
hydroxide. The products were collected by ®ltration. The
yields, physical and spectroscopic date are collected in
Table 10.
8-Bromo-1-(2-chlorophenyl)-3,5-dihydro-4H-2,3-benzodiaz-
epin-4-one (11d). A solution of 10d (4.28 g, 12.1 mmol)
and 98% hydrazine hydrate (1.8 mL) in ethanol (45 mL)
was re¯uxed for 5 h. The mixture was evaporated to
dryness and the residue was dissolved in dichhorometane
(120 mL) and washed with water (3Â20 mL). The organic
layer was dried and evaporated. The crude product was
recystallized from ethanol to give the product (2.26 g).
Yield: 53%. Mp 255±258 ꢀC. C15H10BrClN2O (349.6).
1H NMR: (DMSO-d6): d 3.62 (s, 2H, 5-H), 7.02 (d,
J=1.8 Hz, 1H, 9-H), 7.52 (d, J=8.1 Hz, 1H, 6-H), 7.79
(dd, J1=8.1 Hz, J2=1.8 Hz, 1H, 7-H), 7.55±7.75 (m, 4H,
2-chlorophenyl), 11.35 (br. s, 1H, 3-H).
8-Chloro-1-(4-nitrophenyl)-3-(2-oxopropyl)-3,5-dihydro-
4H-2,3-benzodiazepin-4-one (16). To a solution of 11a
(36.3 g, 115 mmol) in DMF (250 mL) potassium carbon-
ate (19.9 g, 144 mmol) was given and the mixture was
heated at 80 ꢀC for 10 min. Then chloroacetone (11 mL,
138 mmol) was added and the mixture was heated at
80 ꢀC for 2 h. The reaction mixture was then cooled to rt
and poured onto ice (1.2 kg). The precipitate was collected
by ®ltration and washed with water. The crude product
was recrystallized from acetic acid to give 16 (39.3 g).
General method for the synthesis of 3,5-dihydro-4H-2,3-
benzodiazepine-4-thione derivatives 12a±d. To a solution
of the corresponding 4-oxo-2,3-benzodiazepine derivative
(11a±d) (38.0 mmol) in dry pyridine (150 mL) phosphorous
pentasul®de (13.3 g, 60.0 mmol) was given and the mixture
was kept at 80 ꢀC for 2±3 h. The reaction mixture was then
cooled to rt and poured onto ice (1 kg). The precipitate was
collected by ®ltration and washed with water. The crude
products were recrystallized from 2-methoxyethanol.
ꢀ
1
Yield: 92%. Mp 222±224 C. C18H14ClN3O4 (371.8). H
NMR: (DMSO-d6): d 2.12 (s, 3H, CH3), 3.75 (br. s, 2H, 5-
H), 4.72 (s, 2H, CH2), 7.23 (d, J=2.1 Hz, 1H, 9-H), 7.63
(d, J=8.3 Hz, 1H, 6-H), 7.74 (dd, J1=8.3 Hz, J2=2.1Hz,
1H, 7-H), 7.83 (d, J=8.8 Hz, 2H) and 8.33 (d, J=8.8Hz,
2H, nitrophenyl).
8-Chloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-
epine-4-thione (12a). Yield: 71%. Mp 231±234 ꢀC.
C15H10ClN3O2S (331.8). H NMR: (DMSO-d6): d 4.00
(br. s, 2H, 5-H), 7.48 (d, J=8.2 Hz, 1H, 6-H), 7.15 (d,
J=1.9Hz, 1H, 9-H), 7.72 (dd, J1=8.2 Hz, J2=1.9Hz,
1H, 7-H), 7.80 (d, J=8.7 Hz, 2H) and 8.28 (d, J=8.7Hz,
2H, nitrophenyl).
8-Chloro-1-(4-nitrophenyl)-3-phenacyl-3,5-dihydro-4H-
2,3-benzodiazepin-4-one (17). The compound was syn-
thesized from 11a according to the procedure as given
above by using 2-chloroacetophenone. Yield: 83%. Mp
223±225 ꢀC. C23H16ClN3O4 (433.8). 1H NMR: (DMSO-
d6): d 3.8 (br. s, 2H, 5-H), 5.4 (br. s, 2H, phenacyl), 7.22
(d, J=1.9 Hz, 1H, 9-H), 7.64 (d, J=8.3 Hz, 1H, 6-H),
7.74 (dd, J1=8,3 Hz, J2=1.9 Hz, 1H, 7-H), 7.54 (dd,
J1=J2=7.5 Hz, 2H, phenyl), 7.66 (dd, J1=J2=7.5 Hz,
1H, phenyl), 7.98 (d, J=7.5 Hz, 2H, phenyl), 7.83 (d,
J=8.8 Hz, 2H) and 8.32 (d, J=8.8 Hz, 2H, nitrophenyl).
1
8-Bromo-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzodiaz-
epine-4-thione (12b). Yield: 67%. Mp 220±223 ꢀC. C15
H10BrN3O2S (376.8). 1H NMR: (DMSO-d6): d 4.05 (br. s,
2H, 5-H), 7.35 (d, J=1.8 Hz, 1H, 9-H), 7.48 (d, J=8.3Hz,
1H, 6-H), 7.88 (dd, J1=8.3Hz, J2=1.8 Hz, 1H, 7-H), 7.84
(d, J=8.8Hz, 2H) and 8.36 (d, J=8.8Hz, 2H, nitrophe-
nyl), 13.1 (s, 1H, N-H).
8-Chloro-2-methyl-6-(4-nitrophenyl)-11H-imidazolo[1,2-c]
[2,3]benzodiazepine hydrochloride (15b). (Alternative
route.) To a solution of ammonium acetate (180 g) in
acetic acid (180mL) 16 (9.0g, 24.2mmol) was added and
the mixture was heated at 140 ꢀC for 3 h. The reaction
mixture was then cooled to rt and poured onto ice
(0.9 kg). The precipitate was collected by ®ltration and
washed with water. The crude product was recrystallized
twice from DMF to give the product as base (3.42 g).
Yield: 40%. Mp 246±250 ꢀC. A sample was converted to
the hydrochloride with mp 230±231 ꢀC which was iden-
tical with the substance prepared by the general method
described earlier, for further data see Table 10.
7,8-Dichloro-1-(4-nitrophenyl)-3,5-dihydro-4H-2,3-benzo-
diazepine-4-thione (12c). Yield: 61%. Mp 210±213 ꢀC.
C15H9Cl2N3O2S (366.2). MS(EI): m/z: M: 366/368.
8-Bromo-1-(2-chlorophenyl)-3,5-dihydro-4H-2,3-benzodiaz-
epine-4-thione (12d). Yield: 79%. Mp 198±20 ꢀC. C15H10
BrClN2S (365.7). MS(EI): m/z: M: 365/367.
General procedure for the synthesis of 11H-imidazolo
[1,2-c][2,3]-benzodiazepine derivatives (15a±g, 15i±n, 3p,
q). A mixture of the appropriate 2,3-benzodiazepine-4-
thione derivative (12a±d) (10.0 mmol), the correspond-
ing aminoacetal (13a±g) (20.0 mmol) and red mercury
oxide (10.0 mmol) in 2-methoxyethanol was stirred and
heated at re¯ux for 1±10 h. After ®ltration the solvent was
8-Chloro-6-(4-nitrophenyl)-2-phenyl-11H-imidazolo[1,2-c]
[2,3]benzodiazepine (15h). The compound was prepared
from 17 according to the procedure as described above
for 15b. (For yield, physical and spectroscopic data see
Table 10.)

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2138
Table 10. Physical and spectroscopic data of the 11H-imidazolo[1,2-c][2,3]benzodiazepine derivatives 15a±n, 3p±q
Compounda
mp
(ꢀC)
Yield
(%)
Molecular formula
(Mol. mass)
Spectroscopic data:
1H NMR (in DMSO-d6; d) or MS
15 a
210±215 48
229±230 79
205±208 41
207±210 46
C
17H12Cl2N4O2 (375.2)
4.55 (s, 2H, 11-H), 7.22 (d, J=1.8 Hz, 1H, 7-H), 7.65 (d, J=8.2 Hz, 1H, 10-H), 7.84
(dd, J1=8.2 Hz, J2=1.8 Hz, 1H, 9-H), 7.62 (overlapping) and 7.95 (2-H and 3-H), 8.00
(d, J=8.5 Hz, 2H) and 8.43 (d, J=8.5 Hz, 2H, nitrophenyl).
2.25 (d, J=1.0 Hz, 3H, 2-CH3), 4.52 (s, 2H, 11-H), 7.23 (d, J=1.8 Hz, 1H, 7-H), 7.63
(d, J=8.2 Hz, 1H, 10-H), 7.72 (q, J=1.0 Hz, 1H, 3-H), 7.82 (dd, J1=8.2 Hz, J2=1.8 Hz,
1H, 9-H). 7.96 (d, J=8.6 Hz) and 8.44 d, J=8.6 Hz, nitrophenyl).
15 b
15 c
15 d
C
C
C
C
18H14Cl2N4O2(389.2)
18H14Cl2N4O2(389.2) 2.40 (d, J=1.0 Hz 3H, 3-CH3), 7.22 (d, J=1. Hz, 1H, 7-H), 7.42 (q, J=1.0 Hz, 1H, 2-H),
7.62 (d, J=8.2 Hz, 1H, 10-H), 7.86 (dd, J1=8.2 Hz, J2=1.8 Hz, 1H, 9-H),
8.05 (d, J=8.6 Hz) and 8.40 (d, J=8.6 Hz, nitrophenyl).
19H16Cl2N4O2(403.3)
2.22 (s, 3H), 2.35 (s, 3H), 4.49 (s, 2H, 11-H), 7.22 (d, J=1.9 Hz, 1H, 7-H), 7.63
(d, J=8.2 Hz, 1H, 10-H), 7.86 (dd, J1=8.2 Hz, J2=1.9 Hz, 1H, 9-H), 8.02 (d, J=8.7 Hz)
and 8.40 (d, J=8.7 Hz, nitrophenyl).
15 e
15 fb
150±153 20
270±272 30
19H16Cl2N4O2(403.3)
MS(EI): m/z: M: 403/405.
C23H14ClN5O4(459.8)
C22H14ClN5O(415.8)
C23H15ClN4O2414.8
4.25 (br. s, 2H, 11-H), 7.26 (d, J=1.8 Hz, 1H, 7-H), 7.55 (s, 1H, 2-H), 7.72 (d, J=8.3 Hz,
1H, 10-H), 7.76 (dd, J1=8.3 Hz, J2=1.8 Hz, 1H, 9-H), 7.95 (d, J=8.9 Hz, 2H), 8.04
(d, J=8.9 Hz, 2H) and 8.36 (d, J=8.9 Hz, 2H), 8.41 (d, J=8.9 Hz, nitrophenyls).
4.10 (br. s, 2H, 11-H), 7.22 (d, J=1.9 Hz, 1H, 7-H), 7.35 (s, 1H, 2-H), 7.47 (d, J=8.3 Hz,
1H, 10-H), 7.56 (d, J=6.2 Hz, 2H, pyridyl), 7.60 (dd, J1=8.3 Hz, J2=1.9 Hz, 1H, 9-H), 7.92
(d, J=8.9 Hz, 2H) and 8.37 (d, J=8.9 Hz, 2H, nitrophenyl), 8.72 (d, J=6.2 Hz, 2H, pyridyl).
4.25 (br. s, 2H, 11-H), 7.21 (br. s, 1H, 7-H), 7.72 (br. s, 2H, 9-H and 10-H), 7.12±7.26
(1H, phenyl, overlapping), 7.37 (dd, J1=12=7.3 Hz, 2H, phenyl), 7.78 (d, J=7.3 Hz, 2H,
phenyl), 8.00 (d, J=8.8 Hz, 2H, nitrophenyl), 8.10 (s, 1H, 3-H), 8.42
(d, J=8.8 Hz, nitrophenyl).
15 gb
15 hb
250±252 30
239±232 82
15 i
15 j
15 k
221±224 13 C18H13Cl3N4O2(423.7)
240±244 41 C18H13Cl3N4O2(423.7)
MS (EI): m/z (%): M 386/388 (100/68), 385/387 (45/30), 339/341 (16/12).
MS (EI): m/z (%): M 386/388 (73/45), 385/387 (100/68), 339/341 (20/14).
215±220 28
194±202 50
212±219 42
C
C
C
18H14BrClN4O2(433.8) 2.25 (d, J=1.0 Hz, 3H, 2-CH3), 4.43 (br. s, 2H, 11-H), 7.35 (d, J=1.9 Hz, 1H, 7-H), 7.58
(d, J=8.3 Hz, 1H, 10-H), 7.71 (q, J=1.0 Hz, 1H, 3-H), 7.96 (dd, J1=8.3 Hz, J2=1.9 Hz,
1H, 9-H), 7.98 (d, J=8.82 Hz) and 8.43 (d, J=8.8 Hz, nitrophenyl).
15 l
18H14BrClN4O2(433.8)
2.42 (d, J=1.0 Hz, 3H, 3-CH3), 4.50 (s, 2H, 11-H), 7.32 (d, J=1.8 Hz, 1H, 7-H), 7.42
(q, J=1.0 Hz, 1H, 2-H), 7.55 (d, J=8.1 Hz, 1H, 10-H), 7.97 (dd, J1=8.1 Hz, J2=1.8 Hz,
1H, 9-H), 8.05 (d, J=8.7 Hz) and 8.42 (d, J=8.7 Hz, nitrophenyl).
2.22 (s, 3H), 2.34 (s, 3H), 4.45 (s, 2H, 11-H), 7.42 (d, J=1.9 Hz, 1H, 7-H), 7.58
(d, J=8.1 Hz, 1H, 10-H), 7.95 (dd, J1=8.1 Hz, J2=1.9 Hz, 1H, 9-H), 8.01
(d, J=8.8 Hz) and 8.42 (d, J=8.8 Hz, nitrophenyl).
15 m
19H16BrClN4O2(447.8)
15 nb
3 pc,d
3 qc,d
Foam
Ð
Ð
60
Ð
Ð
C22H14BrN5O(460.3)
C18H14BrCl2N3(423.2)
C18H14BrCl2N3(423.2)
MS(EI): m/z: M: 460/462.
aHydrochlorides.
bData refer to the base.
cFor Mp and yield see Table 1.
dFor spectroscopic data see Table 12.
8-Chloro-4-methylthio-1-(4-nitrophenyl)-5H-2,3-benzo-
diazepine (18). Thione 12a (3.32 g 10.0 mmol) was dis-
solved in acetone (200mL) and potassium carbonate
(2.76 g 20.0mmol) and methyliodide (1.87 mL, 30.0
mmol) were added. The mixture was stirred at rt for 3 h,
then the product was ®ltered, washed with water and
recrystallized from DMF ꢀto give the title product (2.84 g).
Yield: 82%. Mp 249±252 C. C16H12ClN3O2S (345.8). 1H
NMR: d 2.42 (s, 3H, S-CH3), 3.35 (d, J=13.0 Hz, 5-H),
and 3.45 (d, J=13.0 Hz), 7.20 (d, J=2.0 Hz,1H, 9-H),
7.25 (d, J=8.2Hz, 1H, 6-H), 7.55 (dd, J1=8.2Hz,
J2=2.0Hz, 1H, 7-H), 7.87 (d, J=8.8Hz) and 8.38 (d,
J=8.8Hz, nitrophenyl).
were recrystallized. The yields, physical and analytical
data are collected in Table 11.
7-Chloro-3-methyl-1-(4-nitrophenyl)isochromane (21).
Prepared from 1-(4-chlorophenyl)-2-propanol33 accord-
ing to the general method as described for 9a±d. Yield:
32%. Mp 120±123 ꢀC. C16H14ClNO3 (303.8). 1H NMR: d
1.42 (d, J=6.4 Hz, 3H, 3-CH3), 2.7±2.95 (m, 2H, 4-H),
4.02 (m, 1H, 3-H), 5.80 (s, 1H, 1-H), 6.60 (d, J=2.0Hz,
1H, 8-H), 7.10 (d, J=8.2Hz, 1H, 5-H), 7.16 (dd,
J1=8.2 Hz, J2=2.0 Hz, 1H, 6-H), 7.53 (d, J=8.8 Hz, 2H)
and 8.24 (d, J=8.8 Hz, 2H, nitrophenyl).
7-Chloro-3-methyl-1-(4-nitrophenyl)-2-benzopyrilium per-
chlorate (22). To an ice cold solution of 21 (6.8 g,
22.4 mmol) in acetone (70 mL) Jones reagent (29 mL,
78 mmol) was added dropwise during 1h and the mixture
was then stirred at rt for 4 h. The separated chromium
salt was ®ltered and the solution evaporated to dryness.
The residue was suspended with water (25 mL) and ®l-
tered. The precipitate was then dissolved in hot acetic
acid (76 mL) and 70% perchloric acid (1.48 mL) was
General procedure for the synthesis of 3-substitued 8-
chloro-6-(4-nitrophenyl)-11H-1,2,4-triazolo[4,5-c][2,3]ben-
zodiazepine derivatives (19a±d). 18 (3.45 g 10.0 mmol)
was dissolved in DMF (120 mL) then the corresponding
acylhydrazide (25.0 mmol) and concd HCl (0.5 mL)
were added and the mixture was stirred and heated at
120±130 ꢀC for 9±15 h. The reaction mixture was poured
onto ice and the product ®ltered. The crude products

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
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2139
Table 11. Physical and spectroscopic data of the 3-substitued 8-chloro-6-(4-nitrophenyl)-11H-1,2,4-triazolo[4,5-c][2,3]benzodiazepine derivatives
19a±d
Compound
mp
(ꢀC)
Yield
(%)
Molecular formula
(Mol. mass)
Spectroscopic data:
1H NMR or MS
19 a
19 b
271±274
287±289
72
86
C17H12ClN5O2 (353.8)
2.58 (s,3H) 4.16 (s, 2H), 7.12 (d, J=1.9 Hz, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.55
(dd, J1=8.3 Hz, J2=1.9 Hz, 1H), 7.90 (d, J=8.7 Hz) and 8.35 (d, J=8.7 Hz, nitrophenyl).
C21H13ClN6O2 (416.8) (in DMSO-d6) 4.46 (br. s, 2H), 7.24 (br. s, 1H), 7.78 (br. s, 2H), 7.98 (d, J=8.8 Hz, 2H)
and 8.42 (d, J=8.8 Hz, 2H, nitrophenyl), 8.18 (d, J=5.5 Hz, 2H) and 8.83
(d, J=5.5 Hz, 2H, pyridyl).
19 c
19 d
287±290
266±268
68
88
C22H13ClN6O4(460.8)
18H14ClN5O3(383.8)
MS(EI):m/z: M:460/462.
MS(EI): m/z: M: 383/385.
C
added. A product separated which was ®ltered after
cooling and washed with acetic acid to give the title
product. Yield: 42% (3.73 g). Mp 247±255 ꢀC. C16H11
azodicarboxylate (1.52 mL, 9.7 mmol) in THF (11 mL)
was added dropwise. After stirring for 3 h at rt the sol-
vent was removed and the residue recrystallized from
ethanol to give 26 (1.34 g). Mp 254±256 ꢀC (dec.).
(MS(FAB): M: 458/460.)
1
Cl2NO7 (400.2). IR: 1096 cm (ClO4 ).
8-Chloro-4-methyl-1-(4-nitrophenyl)-5H-2,3-benzodiaze-
pine (23). Compound 22 (4.1g, 10.2 mmol) was added to
a solution of 98% hydrazine hydrate (1.5 mL, 70.7 mmol)
in DMF (20 mL) at 10±15 ꢀC. The reaction mixture was
then stirred for 1.5 h at rt Water (25 mL) was added and
the separated precipitate was collected by ®ltration and
washed with water. The crude product was recrystallized
from isopropanol. Yield: 87% (2.82 g). Mp 199±203 ꢀC.
The solution of the intermediate 26 (1.34 g, 2.9 mmol) and
98% hydrazine hydrate (1.09 mL, 21.7 mmol) in methanol
(134mL) was heated at boiling for 4 h. After evaporation
of the solvent the residue was treated with methanol
(50 mL) and the resulting precipitate was ®ltered. The ®l-
trate was then evaporated and the residue suspended with
water to give 27 (0.97 g, mp 105±107 ꢀC (dec.)). This crude
intermediate was taken up with acetic anhydride (8 mL)
and stirred for 2 h. Dilution with water (40 mL) gave a
solid substance which was collected by ®ltration. Column
chromatography of this product with the eluent ethyl
acetate:benzene (4:1) gave the acetylamino derivative 28
(0.60 g). Mp 216±218 ꢀC. (MS(FAB): M: 370/372.)
1
C16H12ClN3O2 (313.7). H NMR: (DMSO-d6): d 2.11 (s,
3H, CH3), 2.93 and 3.65 (d, J=12.2 Hz, 2H, 5-H), 7.33 (d,
J=2.0Hz, 1H, 9-H), 7.61 (d, J=8.2 Hz, 1H, 6-H), 7.73
(dd, J1=8.2Hz, J2=2.0 Hz, 1H, 7-H), 7.82 (d, J=8.7Hz,
2H), 8.32 (d, J=8.7Hz, 2H).
8-Chloro-4-formyl-1-(4-nitrophenyl)-5H-2,3-benzodiaze-
pine (24). To a solution of 23 (9.17 g, 29.0 mmol) in diox-
ane (120 mL) powdered selenium dioxide (2.27 g,
20.5 mmol) was given and the mixture was stirred at 90ꢀC
for 40min. Charcoal was added to the mixture and it was
®ltered. The clear solution was poured into water (1.5 L)
and the separated crystalls were ®ltered and washed with
water. Column chromatography with benzene as eluent
gave the aldehyde. Yield: 29% (2.8g). Mp 208±210 ꢀC.
A solution of 28 (0.59 g, 1.6 mmol) and POCl3 (0.73 mL,
7.95 mmol) in 1,2-dichloroethane (30 mL) was heated at
re¯ux for 3 h. The solution was then chilled with ice and
treated with sodium hydrogencarbonate solution. After
separation the organic phase was washed with water,
dried and evaporated. The residual oil was chromato-
graphed with an eluent of ethyl acetate:benzene (4:1) to
give 8-chloro-3-methyl-6-(4-nitrophenyl)-11H-imidazolo
[3,4-c][2,3]benzodiazepine (29), which was then reduced
according to the general procedure as described for
compounds 3a±n. The crude product was boiled with
ethanol to remove impurities. By the above sequence
0.12 g of pure 5 could be prepared. Mp 256±258 ꢀC
(dec.). C18H15ClN4 (322.8). MS(EI): m/z (%): M: 322/
324 (100/33), 321/323 (26/9), 108.5 (22), 65 (11), 219
(10), 106 (10), 218 (8), 217 (8).
1
C16H10ClN3O3 (327.7). H NMR: (DMSO-d6): 2.98 (d,
J=13.1 Hz) and 4.13 (d, J=13.1 Hz, 2H, 5-H), 7.42 (d,
J=2.2Hz, 1H, 9-H), 7.50 (d, J=8.3 Hz, 1H, 6-H), 7.73
(dd, J1=8.3Hz, J2=2.2 Hz, 1H, 7-H), 7.92 (d, J=8.9Hz,
2H) and 8.36 (d, J=8.9 Hz, 2H, nitrophenyl).
6-(4-Aminophenyl)-8-chloro-3-methyl-11H-imidazolo[3,4-c]
[2,3]benzodiazepine (5). The solution of 24 (2.15 g,
6.60mmol) in a 1:1 mixture of THF:water (88 mL) was
chilled with icewater and sodium borohydride (0.12 g
3.30mmol) was given in portions. After stirring at rt for
40min the solution was diluted with water (90 mL) to give
a precipitate which was ®ltered and after drying chroma-
tographed with the eluent of benzene:ethyl acetate (1:1) to
give pure 4-(hydroxymethyl)-8-chloro-1-(4-nitrophenyl)-
5H-2,3-benzodiazepine (25; 1.62 g). Mp >163ꢀC (decom-
position). (MS(FAB): M: 329/331.)
6-(4-Aminophenyl)-8-chloro-2-ethoxycarbonyl-11H-pyr-
rolo[1,2-c][2,3]benzodiazepine (6). The solution of 23
(0.50 g, 1.60 mmol) and ethyl bromopyruvate (0.27 mL,
2.20 mmol) in ethanol (20 mL) was heated at re¯ux for
12 h. After evaporation of the solvent the residue was
chromatographed with benzene as eluent to give 30
(0.29 g) which was reduced without further puri®cation
according to the general procedure as described for 3a±
n. It was recrystallized from ethanol (0.11 g). Mp 247±
249 ꢀC. C21H18ClN3O2 (379.8). MS (EI): m/z (%): M:
379/381 (100/35), 378/380 (41/14), 350/352 (35/14), 306/
308 (35/12), 65 (23).
The intermediate 25 (1.62 g, 4.9 mmol), triphenylphosphine
(2.54 g, 9.7 mmol) and phthalimide (1.42 g, 9.7 mmol) were
dissolved in dry THF (72 mL) and a solution of diethyl

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
2140
General procedure for the reduction of the nitro compounds
15a±n, 19a±d, 29 and 30. (Preparation of 3a±n, 4a±d, 5
and 6). To a stirred solution of the corresponding nitro
compound (2mmol) in a 1:1 mixture of methanol and di-
chloromethane hydrazine hydrate (4±5 equiv) and RaNi
(0.1±2g) were added. After stirring at 20±40 ꢀC for 1±5 h
the catalyst was ®ltered and the solvents evaporated. The
residues were recrystallized from ethanol. Mp-s and yields
of the aminophenyl compounds 3a±n and 4a±d are shown
in Table 1 and 2 respectively. The composition and spec-
troscopic data of 3a±3r and 4a±d are collected in Table 12.
Electrophysiological studies. Electrophysiological experi-
ments were carried out on acutely isolated cerebellar
Purkinje cells according to Bleakman et al.19 Brie¯y:
cells were isolated from the cerebellum of 6±9-day-old
rats using enzymatic treatment and mechanical tritura-
tion, then plated to poly-l-lysine coated glass coverslips.
Cells were kept alive in a tissue culture medium, in a
CO2 thermostat, and used for electrophysiological
experiments on the day of isolation. Purkinje cells
(identi®ed by their bigger size) were patched with glass
electrodes (4±6 Mꢀ). The membrane potential was ®xed
at 70 mV. Experiments were performed at room tem-
perature. The internal recording solution contained NaCl
(140mM), MgCl2 (1mM), HEPES (10mM), glucose
(10 mM), EGTA (0.1 mM), pH=7.2. The cells were
continuously perfused with a solution composed of
NaCl (138 mM), KCl (5 mM), CaCl2 (5 mM), MgCl2
(1 mM), HEPES (10 mM), glucose (10 mM), pH=7.35.
Excitatory amino acids (kainate 100 mM or S-AMPA
5 mM) were applied alone, or in combination with various
concentrations of 1, 2 or 3b. The percentage decrease of
the plateau current was evaluated. IC50ÆS.E.M. values
were calculated from 4±6 concentrations (5±6 cells for
each concentration), using the microcal origin 4.1
computer program for sigmoidal curve ®tting.
6-(4-Acetylaminophenyl)-8-chloro-2-methyl-11H-imida-
zolo[1,2-c][2,3]benzodiazepine (3o). Prepared from 3b
(0.46 g, 1.42 mmol) in pyridine (8 mL) by reacting with
acetylchloride (0.20 mL) at 5±10 ꢀC for 1.5 h. Dilution
with ice water gave a solid precipitate which was
recrystallized from ethanol. For yield and mp see Table
1, spectroscopic data are shown in Table 12.
8-Chloro-3-methyl-6-phenyl-11H-imidazolo[1,2-c][2,3]ben-
zodiazepine (3r). To a stirred solution of 3c (1.10 g,
3.20mmol) in DMF (12 mL) at 65 ꢀC isoamyl nitrite
(0.80 mL) was added droppwise over 8 min. Heating was
continued for 30min and the cooled mixture was then
treated with 5N HCl. The product was extracted with
ether and puri®ed by column chromatography. Eluent:
chloroform:methanol (98:2). For yield and mp see Table
1, spectroscopic data are shown in Table 12.
In vivo experiments
Animals were purchased from Charles River Hungary Ltd.
(Budapest, Hungary) and were housed for a minimum of
six days prior to experiments with free access to stan-
dard laboratory diet and tap water and maintained on a
12±12 h light-dark cycle (light from 6.00 am to 6.00
p.m.). All compounds under study were suspended in
saline (ip administration) or distilled water (oral
administration) containing 1±2% Tween-80. Control
animals received vehicle under the same conditions.
ED50 and LD50 values were calculated by the Litch®eld±
Wilcoxon method.34 The volumes of administration
were 0.1 mL/10 g and 0.5 mL/100 g body weight for mice
and rats, respectively, unless otherwise stated.
Biology, in vitro methods
Retinal spreading depression (S.D.) test. Experiments
were performed according to Sheardown.17 The posterior
chamber of each eye of 1±5-day-old chickens was dis-
sected and placed in a Petri dish containing physiological
saline of the following composition: NaCl (100mM), KCl
(6mM), CaCl2 (1 mM), MgSO4 (1 mM), NaHCO3
(30 mM), NaH2PO4 (1mM). The solution was saturated
with 95% O2 and 5% CO2 and maintained at 26 ꢀC. The
eyes were initially incubated in normal saline for 30 min
and then transferred to a solution containing AMPA
(5 mM) or kainate (5 mM). These solutions triggered
S.D., which could be easily observed by eye. The latency
of S.D. was determined. The presence of a white area
(0.5 mm in diameter ) was taken as the onset of S.D. The
eye cups were then returned to normal saline. After a fur-
ther 15 min recovery period the eyes were incubated in a
solution containing the test compounds and incubated
for 15min. Thereafter the eyes were transferred to a glu-
tamate agonist containing solution which also contained
the test compound and the latency of S.D. was determined
again. 60min following this measurement, the whole pro-
cedure was repeated, with another test substance/con-
centration. Each drug concentration was tested in 6 retina
preparations. An increase in the latency of 30 s or more
was considered to be 100% inhibition of S.D. (cut-o€ time
was 1 min).17 The drug e€ects therefore are expressed as
the percentage maximum inhibition obtained for a given
concentration. Dose-response curves were constructed
from 3±6 concentration points and IC50ÆS.E.M. values
were calculated by sigmoidal curve ®tting using the
microcal origin 4.1 computer program.
Behavioural changes in mice. 5 male CD1 mice (21±26 g)
were starved for 16 h and treated with test compounds in
doses of 100 and 200 mg/kg ip and po, respectively. The
gross behavioural changes were evaluated continuously
for 5 h according to Irwin.35
Seizure assays. 10 male CD1 mice (21±26 g) per group,
after 16 h starvation, were used. Animals were treated
orally with test compounds 60 min before maximal elec-
troshock (MES)36 or chemical convulsants (metrazole
130 mg/kg ip, strychnine 3 mg/kg ip, bemegride 50 mg/
kg ip, bicuculline 1 mg/kg iv, nicotine 3.5 mg/kg iv, 4-
aminopyridine 12.5 mg/kg ip and 3-mercapto-propionic
acid 110 mg/kg iv).37 Prevention of tonic convulsion
(MES) and/or death (convulsive agents) served as measure
of anticonvulsive potency.
Muscle relaxation in mice. (Inclined screen and rotarod
tests). For determination of muscle relaxant activity the
inclined screen test38 and rotarod test39 were used. 10
male CD1 mice (21±25 g) per group were examined.

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
Â
2141
Table 12. Composition and spectroscopic data of compounds 3a±r and 4a±d
Compound
Molecular formula
(Mol. mass)
Spectroscopic data:
1H NMR (in DMSO-d6; ) or MS
3a
C17H13ClN4 (308.8)
4.00 (br. s, 2H, 11-H), 5.85 (br. s, NH2), 6.84 and 7.38 (d, J=1.5 Hz, 2-H and 3-H), 6.63
(d, J=8.7 Hz, 2H) and 7.36 (d, J=8.7 Hz, 2H, aminophenyl), 7.21 (d, J=2.0 Hz, 1H, 7-H), 7.58
(d, J=8.3 Hz, 1H, 10-H), 7.63 (dd, J1=8.3 Hz, J2=2.0 Hz, 1H, 9-H).
3b
3c
3d
3e
3f
C18H15ClN4(322.8)
2.04 (d, J=1.1 Hz, 1H, 2-CH3), 3.91 (br. s, 2H, 11-H), 5.80 (br. s, 2H, NH2), 6.63 (d, J=8.7 Hz, 2H)
and 7.37 (d, J=8.7 Hz, 2H, aminophenyl), 7.07 (q, J=1.1 Hz, 1H, 3-H), 7.19 (d, J=2.1 Hz, 1H, 7-H),
7.56 (d, J=8.3 Hz, 2H, 10-H), 7.64 (dd, J1=8.3 Hz, J2=2.1 Hz, 1H, 9-H).
(in CDCl3) 2.32 (d, J=1.0 Hz, 3H, 3-CH3), 3.92 (br. s, 2H, 11-H), 4.08 (br. s, 2H, NH2), 6.63
(q, J=1.0 Hz, 1H, 2-H), 6.75 (d, J=8.5 Hz, 2H) and 7.55 (d, J=8.5 Hz, 2H, aminophenyl), 7.29
(d, J=1.9 Hz, 1H, 7-H), 7.36 (d, J=8.1 Hz, 1H, 10-H), 7.44 (dd, J1=8.1 Hz, J2=1.9 Hz, 1H, 9-H).
2.00 and 2.18 (s, 3H, CH3), 3.90 (br. s, 2H, 11-H), 5.82 (br. s, 2H, NH2), 6.68 (d, J=8.8 Hz, 2H)
and 7.42 (d, J=8.8 Hz, 2H, aminophenyl), 7.20 (d, J=1.9 Hz, 1H, 7-H), 7.54 (d, J=8.3 Hz, 1H, 10-H),
7.64 (dd, J1=8.3 Hz, J2=1.9 Hz, 1H, 9-H).
C18H15ClN4(322.8)
C19H17ClN4(336.8)
C19H17ClN4(336.8)
C23H18ClN5(399.9)
(in CDCl3) 1.26 (t, J=7.3 Hz, 3H), 2.6 (q, J=7.3 Hz, 2H), 3.94 (br. s, 2H, 11-H), 6.72 (d, J=8.6 Hz, 2H)
and 7.52 (d, J=8.6 Hz, 2H, aminophenyl), 6.97 (s, 1H, 3-H), 7.27 (d, J=2.0 Hz, 1H, 7-H), 7.34
(d, J=8.2 Hz, 1H, 10-H), 7.45 (dd, J1=8.2 Hz, J2=2.0 Hz, 1H, 9-H).
3.6-4.3 (2H, 11-H), 5.25 (br. s, 2H) and 5.86 (br. s, 2H, NH2), 6.64 (d, J=8.8 Hz, 2H), 6.66
(d, J=8.8 Hz, 2H) and 7.27 (d, J=8.8 Hz, 2H), 7.35 (d, J=8.8 Hz, 2H, aminophenyls), 7.30
(d, J=1.9 Hz, 1H, 7-H), 7.60 (d, J=8.3 Hz, 1H, 10-H), 7.65 (dd, J1=8.3 Hz, J2=1.9 Hz, 1H, 9-H).
3.8-4.3 (2H, 11-H), 7.30 (d, J=2.0 Hz, 1H, 7-H), 7.46 (s, 1H, 2-H), 7.62 (d, J=8.2 Hz, 1H, 10-H),
7.67 (dd, J1=8.2 Hz, J2=2.0 Hz, 1H, 9-H), 6.68 (d, J=8.6 Hz, 2H) and 7.40 (d, J=8.6 Hz, 2H,
aminophenyl), 7.68 (d, J=5.6 Hz, 2H) and 8.62 (d, J=5.6 Hz, 2H, pyridyl).
MS (EI): m/z (%): M: 384/386 (100/33).
(in CDCl3) 2.22 (d, J=1.0 Hz, 3H, 2-CH3), 3.96 (br. s, 2H, 11-H), 4.08 (br. s, 2H, NH2), 6.98
(q, J=1.0 Hz, 1H, 3-H), 6.65 (d, J=8.0 Hz, 2H) and 7.53 (d, J=8.6 Hz, 2H,
aminophenyl), 7.30 (s, 1H), 7.39 (s, 1H).
3g
C22H16ClN5(385.9)
3h
3i
C23H17ClN4(384.9)
C18H14Cl2N4(357.2)
3j
C
18H14Cl2N4(357.2)
2.21 (d, J=1.0 Hz, 3H, 3-CH3), 4.00 (br. s, 2H, 11-H), 6.60 (q, J=1.0 Hz, 1H, 2-H), 6.68
(d, J=8.6 Hz, 2H) and 7.45 (d, J=8.6 Hz, 2H, aminophenyl), 7.40 (s, 1H), 7.93 (s, 1H).
2.02 (d, J=1.2 Hz, 3H, 2-CH3), 3.90 (br. s, 2H, 11-H), 5.90 (br. s, 2H, NH2), 7.07 (q, J=1.2 Hz,
1H, 3-H), 6.62 (d, J=8.5 Hz, 2H) and 7.35 (d, J=8.5 Hz, 2H, aminophenyl), 7.32 (d, J=2.2 Hz,
1H, 7-H), 7.47 (d, J=8.3 Hz, 1H, 10-H), 7.75 (dd, J1=8.2 Hz, J2=2.2 Hz, 1H, 9-H).
2.22 (d, J=1.0 Hz, 3H, 3-CH3), 3.92 (br. s, 2H, 11-H), 5.82 (br. s, 2H, NH2), 6.55 (q, J=1.0 Hz,
1H, 2-H), 6.65 (d, J=8.6 Hz, 2H) and 7.42 (d, J=8.6 Hz, 2H, aminophenyl), 7.32 (d, J=1.8 Hz,
1H, 7-H), 7.50 (d, J=8.0 Hz, 1H, 10-H), 7.72 (dd, J1=8.0 Hz, J2=1.8 Hz, 1H, 9-H).
1.98 (s, 3H) and 2.16 (s, 3H, CH3), 3.85 (br. s, 2H, 11-H), 5.80 (s, 2H, NH2), 6.65 (d, J=8.6 Hz, 2H)
and 7.34 (d, J=8.6 Hz, 2H, aminophenyl), 7.31 (d, J=1.9 Hz, 1H, 7-H), 7.45 (d, J=8.2 Hz, 1H,
10-H), 7.75 (dd, J1=8.2 Hz, J2=1.9 Hz, 1H, 9-H).
3.7±4.3 (br, 2H, 11-H), 5.95 (br. s, 2H, NH2), 6.67 (d, J=8.6 Hz, 2H) and 7.40 (d, J=8.6 Hz, 2H,
aminophenyl), 7.47 (s, 3H, 2-H), 7.45 (d, J=2.0 Hz, 1H, 7-H), 7.57 (d, J=8.2 Hz, 1H, 10-H), 7.78
(dd, J1=8.2 Hz, J2=2.0 Hz, 1H, 9-H), 7.71 (d, J=5.6 Hz, 2H) and 8.63 (d, J=5.6 Hz, 2H, pyridyl).
(in CDCl3) 2.18 (d, J=1.0 Hz, 3H, 2-CH3), 2.23 (s, 3H, acetyl), 3.94 (br. s, 2H, 11-H), 6.98
(q, J=1.0 Hz, 1H, 3-H), 7.18 (d, J=2.2 Hz, 1H, 7-H), 7.31 (d, J=8.2 Hz, 1H, 10-H), 7.46
(dd, J1=8.2 Hz, J2=1.9 Hz, 1H, 9-H), 7.63 and 7.68 (d, J=8.8 Hz,
3k
C18H15BrN4(367.3)
3l
C18H15BrN4(367.3)
3m
3n
3o
C19H17BrN4(381.3)
C22H16BrN5(430.3)
C20H17ClN4O(364.8)
acetylaminophenyl), 8.13 (br. s, 1H, NH-Ac).
2.32 (d, J=1.0 Hz, 3H, 2-CH3), 4.48 (br. s, 2H, 11-H), 7.12 (q, J=1.0 Hz, 1H, 3-H),
7.55±7.80 (m, 5H), 7.90±8.00 (m, 2H).
2.35 (d, J=1.0 Hz, 3H, 3-CH3), 4.45 (br. s, 2H, 11-H), 7.08 (d, J=2.1 Hz, 1H, 7-H), 7.43
(q, J=1.0 Hz, 1H, 2-H), 7.58 (d, J=8.3 Hz, 1H, 10-H), 7.60±7.73 (m, 4H), 7.95
(dd, J1=8.3 Hz, J2=2.1 Hz, 1H, 9-H).
3p a
3q a
C18H14BrCl2N3(423.1)
C18H14BrCl2N3(423.1)
3r
4a
4b
4c
C18H14ClN3(307.8)
C17H14ClN5(323.8)
C21H15ClN6(386.8)
2.30 (br. s, 3H, CH3), 4.05 (br. s, 2H, 11-H), 6.63 (br. s, 1H, 2-H), 7.12 (d, J=1.9 Hz,
1H, 7-H), 7.50±7.85 (m, 7H).
2.40 (br. s, 3H, 3-CH3), 4.13 (br, 2H, 11-H), 5.94 (br, 2H, NH2), 6.64 (d, J=8.6 Hz, 2H) and 7.43
(d, J=8.6 Hz, 2H, aminophenyl), 7.22 (br. s, 1H, 7-H), 7.65 (br. s, 2H, 9-H and 10-H).
3.95±4.6 (br, 2H, 11-H), 6.67 (d, J=8.6 Hz, 2H) and 7.42 (d, J=8.6 Hz, 2H, aminophenyl), 7.28
(br. s, 1H, 7-H), 7.68 (br. s, 2H, 9-H and 10-H), 8.02 (br. s, 2H, pyridyl), 8.82 (br. s, 2H, pyridyl).
(in CDCl3) 4.12 (br, 2H, 11-H), 6.72 (d, J=8.6 Hz, 2H), 6.79 (d, J=8.6 Hz, 2H) and 7.54
(d, J=8.6 Hz, 2H), 7.84 (d, J=8.6 Hz, 2H, aminophenyls), 7.32 (d, J=2.1 Hz, 1H, 7-H), 7.40
(d, J=8.2 Hz, 1H, 10-H), 7.50 (dd, J1=8.2 Hz, J2=2.1 Hz, 1H, 9-H)
C22H17ClN6(400.9)
4d
C18H16ClN5O(353.8)
3.35 (s, 3H, OCH3), 4.20 (br. s, 2H, 11-H), 4.62 (s, 2H, O-CH2), 5.98 (br. s, 2H, NH2), 6.63
(d, J=8.7 Hz, 2H) and 7.42 (d, J=8.7 Hz, 2H, aminophenyl), 7.22 (br. s, 1H, 7-H),
7.62 (br. s, 2H, 9-H and 10-H).
aHydrochloride salts.
They were treated ip 30 min before testing. In the
inclined screen test the untrained mice were placed on a
screen inclining 60ꢀ. The number of falling mice was
noted within 60 s test period and scored as positive. In
the rotarod test mice were trained to do coordinated
motor movements for 120 s on the rod (3.5 cm diameter)
rotating at 15 rpm. The impairment of coordinated
motor movements was de®ned as inability of selected
mice to remain on the rotating rod for 120 s test period.
Transient occlusion of the middle cerebral artery (MCA)
in rats. Male CD RB rats, weighing 370±410 g, were

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G. Abraham et al. / Bioorg. Med. Chem. 8 (2000) 2127±2143
2142
used. The anaesthesia was induced by 5% and maintained
by 2% halothane in a mixture of 30% N2O and 70%
O2. The rectal body temperature was controlled and
kept at 37 ꢀC throughout the experimental period using
a thermostatically controlled blanket.
HPLC mobile phase containing a-methyl-dopamine as
internal standard. The samples were centrifuged (10,000Âg
for 15min at 4 ꢀC) and a 5 ml aliquots were injected into the
HPLC system. Calculated values are expressed as mg/g wet
tissue.
The occlusion of the MCA was achieved as follows:22,23
the left common carotid artery (CCA), the extracranial
external carotid artery (ECA) and internal carotid
artery (ICA) were exposed through a midline incision.
The branches of the ECA were then isolated and coa-
gulated. The pterygopalatine artery, this posteriorly
directed extracranial branch of the ICA, was occluded
by a microvascular clip. Next a silk suture was tied
loosely around the mobilised ECA stump which was cut
at its ®rst branching (leaving a stump about 2±3 mm)
and used to introduce the embolus. The CCA was
occluded by microvascular clip and a 5 cm length of 4±0
mono®lament nylon suture, its tip rounded by heating
near a ¯ame, was introduced into the ECA lumen
(22 mm distal to the carotid bifurcation). This was
secured in place by tightening the silk ligature. At this
point the intraluminal suture has blocked the origin of the
MCA occluding of blood ¯ow from the ICA, anterior and
posterior cerebral arteries.
The concentrations of dopamine, DOPAC, HVA, 5-HT
and 5-HIAA in the samples were determined by HPLC/
electrochemistry according to Patthy and Gyenge.41 The
HPLC system used in this study consisted of a Knauer
64 isocratic pump with a pulse dampener, a BAS Unijet
microbore injector equipped with a 5 ml sample loop, a
BAS microbore ODS 3 mm (100Â1 mm) analytical column.
For electrochemical detection, glassy carbon electrodes
(BAS LC-4C) set at +0.75 V against an Ag/AgCl
reference electrode were used. The mobile phase com-
position was aqueous bu€er:acetonitrile (95:5) (v/v). The
aqueous bu€er consisted of 3 mM HFBA (hepta-
¯uorobutyric acid) as pairing ion, 100 mM sodium
hydroxide, 0.1 g/l Na2EDTA, and the pH was adjusted
to 4.3 with 42.5% (w/v) phosphoric acid. All other che-
micals used were of analytical reagent grade. One-way
analysis of variance (ANOVA) followed by the Duncan's
multiple range test was used for statistical analysis.
Acute toxicity in mice. The LD50 values were calculated
from lethality within 14 days after ip and po adminis-
tration of the drug.42
60 min after embolization a second short and light
anaesthesia was induced by face mask. The nylon suture
and the microvascular clip on the CCA were carefully
removed, so the blood-¯ow from the CCA into the ICA
was restored. 24 h after reperfusion the rats were decapi-
tated. Their brains were rapidly removed 5 mm from the
frontal pole 3 coronal sections (2 mm thick) were cut out
from the cerebrum, immersed in a 2% solution of TTC
for vital staining and placed in a 37 ꢀC water-bath for 10
minutes and ®xed in 10% formalin solution. TTC
stained the intact areas of brain to deep red colour, but
did not stain the infarcted tissue.
Acknowledgements
The authors wish to express their thanks to the Hun-
garian National Committee for Technological Develop-
ment (OMFB) for the generous ®nancial support of the
project.
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