Synthesis and evaluation of pharmacological and pharmacokinetic properties of 11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones

Article abstract of DOI:10.1021/jm001012y

A series of 2,3-benzodiazepine derivatives has been previously described as noncompetitive AMPA-type glutamate receptor antagonists potentially useful for treatment of epilepsy. To further explore the structure-activity relationships of AMPA antagonists,

Full text of DOI:10.1021/jm001012y

4834
J . Med. Chem. 2000, 43, 4834-4839
Syn th esis a n d Eva lu a tion of P h a r m a cologica l a n d P h a r m a cok in etic P r op er ties
of 11H-[1,2,4]Tr ia zolo[4,5-c][2,3]ben zod ia zep in -3(2H)-on es
Maria Zappala`,# Rosaria Gitto,# Francesca Bevacqua,# Silvana Quartarone,# Alba Chimirri,*,# Milena Rizzo,‡
Giovambattista De Sarro,§ and Angela De Sarro†
Dipartimento Farmaco-Chimico and Istituto di Farmacologia, Facolta` di Medicina e Chirurgia, Universita` di Messina,
Messina, Italy, and Dipartimento di Scienze Farmacobiologiche and Dipartimento di Medicina Sperimentale e Clinica,
Universita` di Catanzaro, Catanzaro, Italy
Received J une 22, 2000
A series of 2,3-benzodiazepine derivatives has been previously described as noncompetitive
AMPA-type glutamate receptor antagonists potentially useful for treatment of epilepsy. To
further explore the structure-activity relationships of AMPA antagonists, a series of 11H-
[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones (6) was synthesized starting from the cor-
responding bicyclic 1-aryl-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-ones (2, CFM). The
new compounds were found to possess anticonvulsant effects against seizures induced both by
means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock
in Swiss mice. In addition, they antagonize the AMPA-induced seizures, and their anticon-
vulsant activity is reversed by pretreatment with aniracetam, thus suggesting the involvement
of AMPA receptors. The pharmacological studies revealed that the 11H-[1,2,4]triazolo[4,5-c]-
[2,3]benzodiazepin-3(2H)-ones (6) herein reported show anticonvulsant activity comparable to
that of their bicyclic precursors. Furthermore, an HPLC study put in evidence that these tricyclic
derivatives 6 were converted in vivo into the corresponding 2, the agents likely to be mainly
responsible for the anticonvulsant properties observed.
In tr od u ction
complex of subunits and not a pentameric complex as
has hitherto been believed.4 There are 14 distinct
ionotropic GluR subunits with a different distribution
in the brain; additionally, some of these subunits are
regulated developmentally in an age-related manner.
AMPA receptor types are composed of combinations of
GluR1-4 subunits, existing as “flip” and “flop” splice
variants, which mediate fast excitatory postsynaptic
Glutamate (Glu) is the major excitatory neurotrans-
mitter in the vertebrate brain and plays an important
role in neuronal activity via different receptor systems.
Glutamate receptors (GluRs) are involved in such
fundamental processes as neuronal development, learn-
ing, and memory. They are also held responsible for the
destruction of neural tissue during ischemic states and
epileptic seizures and may be involved in a variety of
other neurological diseases. This has resulted in an
interest in the development of GluR ligands as research
tools and potential therapeutic agents.1,2
GluRs are divided into metabotropic receptors
(mGluRs) and ionotropic receptors (iGluRs), the latter
consisting of three primary families: N-methyl-D-as-
partic acid (NMDA) receptor family and non-NMDA
receptor families, composed of the kainic acid (KA)
receptor and the 2-amino-3-(3-hydroxy-5-methylisox-
azol-4-yl)propionic acid (AMPA) receptor.3 The iGluRs
are formed by homo- or hetero-oligomeric assemblies of
agonist binding subunits surrounding a central cation-
conducting pore. The binding of Glu induces conforma-
tional changes which open the channel pore and lead
to influx of cations into the postsynaptic cell. At the
present time, iGluRs are supposed to be a tetrameric
potentials by the flux of Na+ and Ca2+ 5
.
Several AMPA receptor antagonists have been re-
ported in the literature and show promise in terms of
their therapeutic potential for the prevention and treat-
ment of a broad range of acute and chronic neurological
diseases.6,7 The AMPA receptor complex has at least
three distinct binding sites at which antagonists can
act: (a) the Glu binding sites for competitive antago-
nists, (b) an allosteric site at which noncompetitive
antagonists can bind, and (c) a polyamine site, within
the ion channel.8
A selective and noncompetitive blockade of the AMPA
receptor was shown by some 2,3-benzodiazepine deriva-
tives: e.g. 1-(4-aminophenyl)-4-methyl-7,8-methylene-
dioxy-5H-2,3-benzodiazepine (1, GYKI 52466) (Chart 1),
which possess potent anticonvulsant properties but lack
hypnotic activity.9-19 In particular, some of our previous
publications14-16 reported chemical and biological stud-
ies of 1-aryl-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodi-
azepin-4-ones 2 (CFM) and thiocarbonyl analogues 3
(Chart 1), which have shown marked antiepileptic
properties in various seizure models and do not bind to
the GABAergic benzodiazepine receptors. Electrophysi-
ological experiments carried out on the lead compound
2f (CFM-2) and some of its derivatives have confirmed
that their anticonvulsant effects, analogous to those of
* To whom correspondence should be addressed: Alba Chimirri,
Dipartimento Farmaco-Chimico, Universita` di Messina, Viale Annun-
ziata, 98168 Messina, Italy. Phone: +39 090 6766412. Fax: +39 090
355613. E-mail: chimirri@pharma.unime.it.
#
Dipartimento Farmaco-Chimico, Universita` di Messina.
‡ Dipartimento di Scienze Farmacobiologiche, Universita` di Catan-
zaro.
§ Dipartimento di Medicina Sperimentale e Clinica, Universita` di
Catanzaro.
† Istituto di Farmacologia, Universita` di Messina.
10.1021/jm001012y CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/16/2000

11H-[1,2,4]Triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4835
Ch a r t 1
Sch em e 1a
a
Reagents: (i) Lawesson’s reagent, dry toluene, 4, 90-120 min;
GYKI 52466, are mediated through the AMPA subtype
of the iGluR complex in a selective and noncompetitive
fashion.
(ii) H2NNHCO2Et, n-BuOH, 4, 24 h; (iii) SnCl2, EtOH, 70 °C, 90
min.
Extensive structure-activity relationship (SARs) stud-
ies, aimed at exploring the effects of structural modifi-
cations of the heptatomic system of derivatives 2 and
at developing AMPA antagonists with increased potency
and selectivity, longer-lasting activity, and improved
pharmacokinetic features, led to the synthesis of two
series of cyclofunctionalized 2,3-benzodiazepines: i.e.
11H-tetrazolo[1,5-c][2,3]benzodiazepines19a 4 and 11H-
[1,2,4]triazolo[4,5-c][2,3]benzodiazepines19b 5 (Chart 1),
which showed weaker anticonvulsant effects than their
parent compounds 2 and 3.
To determine if the lower potency of the compounds
obtained was due to the cyclofunctionalization of the
diazepine ring, to the nature of the fused five-membered
ring, or to the absence of the lactam moiety, we have
now prepared a series of 11H-[1,2,4]triazolo[4,5-c][2,3]-
benzodiazepin-3(2H)-ones 6. The new annelated 2,3-
benzodiazepines have been evaluated as anticonvulsant
agents in various experimental epilepsy models: i.e.
audiogenic seizures in DBA/2 mice and maximal elec-
troshock (MES) and pentylenetetrazole (PTZ)-induced
seizures in Swiss mice. In addition, to confirm that their
anticonvulsant activity is mediated by interaction with
the AMPA receptor system, derivatives 6b,f were also
evaluated against sound-induced seizures with concomi-
tant treatment with aniracetam, a positive allosteric
modulator of AMPA/KA receptors,14 and against AMPA-
induced seizures. Furthermore, the time course of
anticonvulsant activity was also studied to verify if the
new compounds had a longer-lasting anticonvulsant
activity than GYKI 52466 and CFM derivatives. An-
other objective of this study was to gain insight into the
pharmacokinetic properties of these tricyclic benzodi-
azepines and to correlate the plasma concentrations of
the title compounds and their metabolites with the
anticonvulsant effects.
tivated by transformation into the corresponding thio-
carbonyl derivatives 3, by reaction with Lawesson’s
reagent. Compounds 2 and 3 were prepared according
to a multistep synthetic pathway previously described
in our reports.15,16 By refluxing 3,5-dihydro-4H-2,3-
benzodiazepine-4-thiones 3a -d with ethyl carbazate,
11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-
ones 6a -d were synthesized. Aminophenyl-substituted
derivatives 6e,f were prepared by reduction of the
corresponding nitro analogues 6c,d with tin(II) chloride.
The structures of the compounds obtained were sup-
ported by elemental analyses and spectroscopic mea-
surements (1H NMR).
Resu lts a n d Discu ssion
The anticonvulsant properties of 11H-[1,2,4]triazolo-
[4,5-c][2,3]benzodiazepin-3(2H)-ones 6 were evaluated
after intraperitoneal (ip) administration against audio-
genic seizures in DBA/2 mice (Table 1), which has been
considered an excellent animal model for generalized
epilepsy and for screening new anticonvulsant drugs.20
The results were compared with those of GYKI 52466
and bicyclic parent compounds 2.
As can be seen from the data reported in Table 1,
derivatives 6 possess anticonvulsant potency which is
sensitive to the nature of the substituent on the phenyl
ring at C-6. The introduction of a fluorine atom at
position 4′ and an amino group at position 3′ or 4′ affords
derivatives which display anticonvulsant activity com-
parable to (i.e. 6b) or higher (i.e. 6e,f) than that of GYKI
52466 and unsubstituted derivative 6a . In contrast, the
presence of a nitro group reduces activity. Moreover,
compounds 6 generally induce less motor impairment
than GYKI 52466.
A comparison between the biological results of com-
pounds 6a -f and those of the parent bicyclic derivatives
2a -f reveals that the introduction of the triazolone
nucleus on the diazepine skeleton leads to compounds
with comparable or higher anticonvulsant potency than
the corresponding derivatives 2.
Ch em istr y
The 11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-
ones 6 (Scheme 1) were obtained starting from 3,5-
dihydro-4H-2,3-benzodiazepin-4-ones 2, which were ac-

4836 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Zappala` et al.
Ta ble 1. Anticonvulsant Activity of Compounds 1, 2, and 6 Against Audiogenic Seizures in DBA/2 Mice, TD50 Values on Locomotion
Assessed by Rotarod Test, and Relative Lipophilicity (Rm)
ED50 µmol/kga ((95% confidence limits)
TD50, µmol/kga
TIb
compd
clonic phase
tonic phase
locomotor deficit
TD50/ED50
Rm
1c
35.8 (24.4-52.4)
33.9 (26.0-44.2)
78.0 (46.0-132)
25.3 (16.0-40.0)
31.8 (24.8-40.6)
57.2 (41.5-78.8)
76.1 (47.5-122)
142 (87.3-231)
2.1
4.2
ND
ND
ND
2.7
3.8
2.8
2.8
ND
ND
3.8
4.0
-0.368
-0.280
-0.280
-0.181
-0.145
-0.619
-0.647
-0.508
-0.402
-0.402
-0.368
-0.740
-0.837
2a c
2b
2cc
2d c
2ec
2fc
6a
>150
>120
>120
19.3 (16.9-22.0)
>120
>120
18.3 (16.0-20.8)
>150
>150
51.5 (34.1-77.8)
15.0 (9.01-24.0)
44.6 (32.5-61.2)
32.1 (16.3-63.0)
56.4 (39.2-81.1)
74.1 (60.3-91.0)
20.1 (10.5-38.2)
16.1 (11.8-22.0)
12.6 (8.01-19.0)
27.5 (15.4-49.4)
21.8 (14.3-31.9)
43.2 (32.5-57.4)
58.3 (41.3-82.3)
13.1 (8.04-21.3)
10.2 (8.67-11.93)
56.8 (39.3-82.1)
123 (73.8-206)
90.6 (65.1-126)
6b
6c
6d
6e
>150
>150
76.8 (59.4-99.3)
65.8 (46.2-99.8)
6f
a
All data were calculated according to the method of Litchfield and Wilcoxon.29 At least 32 animals were used to calculate each ED50
b
and TD50 value. TI ) therapeutic index and represents the ratio between TD50 and ED50 (from the clonic phase of the audiogenic seizures).
c Ref 15. ND ) not detectable.
Ta ble 2. Anticonvulsant Activity of 1 and 6 Against the MES-
the right the dose-response curves with a pattern of
activity similar to that of 1 and 2.15
Another objective of the present study was to explore
and PTZ-Induced Seizures in Swiss Mice
ED50, µmol/kga ((95% confidence limits)
a possible metabolic pathway of these new tricyclic 2,3-
benzodiazepines and to correlate the plasma concentra-
tions with their anticonvulsant activity. An HPLC
method has been developed to detect the most active
derivative 6f and its metabolites and to determine the
time profile of plasma concentrations of rats treated ip
with 6f. In the chromatogram from a rat plasma sample
obtained after administration of 6f, the peak corre-
sponding to 2f was also observed; the formation of 2f
was monitored (Figure 1) and it was observed that as
6f plasma concentration decreased, the 2f plasma levels
increased, thus suggesting that 6f was metabolically
converted in vivo into 2f. A comparison between the
time course of plasma levels (Figure 1) and that of
anticonvulsant activity (Figure 2) showed that com-
pound 6f had low anticonvulsant activity during the
period of peak plasma concentration (15 min), whereas
maximal effects of 6f were observed from 45-120 min
after ip administration, that is, after biotransformation
into the bicyclic precursor 2f. On the other hand, when
tested at 15 min from administration 6f was more active
than 2f (ED50 27.8 µmol/kg for 6f and ED50 >50 µmol/
kg for 2f) thus suggesting that 6f acts both as active
compound and after biotransformation. This behavior
could also explain its longer-lasting activity compared
to analogue 2f (Figure 2).
compd
MES
PTZ
1b
6a
6b
6d
6e
6f
35.7 (29.3-43.4)
60.5 (45.6-80.3)
43.4 (33.5-56.0)
75.8 (62.4-92.1)
35.1 (26.8-45.8)
30.8 (18.6-51.0)
68.3 (56.2-83.1)
76.5 (51.8-113)
60.5 (45.6-80.3)
90.7 (72.2-114)
58.9 (43.8-79.1)
57.2 (41.5-78.8)
a
All data were calculated according to the method of Litchfield
and Wilcoxon.29 At least 32 animals were used to calculate each
b
ED50 value. Ref 15.
The relative lipophilicity (Rm) of 2,3-benzodiazepines
2 and 6 is summarized in Table 1. Although in both
series the most active compound is also the most
hydrophilic derivative, a direct correlation between
lipophilicity and anticonvulsant activity cannot be
pointed out. In fact, compounds with the same Rm values
(i.e. 2a and 2b; 6b and 6c) show different potencies, thus
suggesting the importance of other parameters.
Compounds 6 were further evaluated against seizures
induced by MES and PTZ in Swiss mice (Table 2). As
shown in Table 2, the tonic extension of the seizures
induced by MES and the clonic phase of the seizures
induced by PTZ were significantly reduced 45 min after
ip administration of compounds 6, with the same rank
order observed in the audiogenic seizures test.
The mechanism of biotransformation has not been
clarified to date. A possible metabolic pathway might
be the cleavage of triazolone ring with subsequent
decarboxylation and hydrolytic processes. Because com-
pound 6f in vivo furnishes the corresponding noncom-
petitive AMPA receptor antagonist 2f, we suggest that
the anticonvulsant effects observed are attributable to
a noncompetitive blockade of the same receptor. In
conclusion, a new series of annelated 2,3-benzodiaze-
pines was prepared and characterized. The synthetic
approach has been demonstrated to be sufficiently
flexible to afford a variety of 6-substituted 11H-[1,2,4]-
triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones (6).
To establish that the anticonvulsant properties of
compounds 6 are mediated by interaction with the
AMPA receptor complex, an additional test against
AMPA-induced seizures in DBA/2 mice was carried out
on the most active derivatives 6b,f. As shown in Table
3, these compounds afforded effective protection against
the clonic and tonic phases of the seizures induced by
intracerebroventricular (icv) administration of AMPA.
Furthermore, the influence of aniracetam, a positive
allosteric modulator of the AMPA/KA receptor,21 on the
anticonvulsant activity of derivatives 6b,f in DBA/2
mice was also tested (Table 3). An icv injection of
aniracetam (50 nmol/mouse) on its own had no convul-
sant activity. The administration of aniracetam 30 min
before the injection of the tested compounds reduced the
anticonvulsant effects of compounds 6b,f and shifted to
In addition, the present study demonstrated that
compounds 6 are slightly more potent and slightly less
toxic but have a significant improvement in terms of

11H-[1,2,4]Triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4837
Ta ble 3. ED50 Values of 1 and 6b,f Against AMPA-Induced Seizures and Against Audiogenic Seizures after Pretreatment with
Aniracetam in DBA/2 Mice
ED50, µmol/kga ((95% confidence limits)
AMPAb
pretreatment with aniracetamc
compd
clonic phase
tonic phase
clonic phase
tonic phase
1d
6b
6f
57.5 (43.5-76.0)
42.1 (27.5-64.3)
22.1 (13.8-37.2)
40.5 (26.3-60.8)
32.2 (20.6-50.3)
18.1 (11.4-28.7)
134 (88.8-203)*
50.9 (28.3-91.5)
38.4 (20.5-71.9)*
100 (63.4-158)*
38.4 (23.5-62.7)*
24.1 (12.7-45.5)*
a
All data were calculated according to the method of Litchfield and Wilcoxon.29 At least 32 animals were used to calculate each ED50
b
value. AMPA was administered icv at the CD97 for either clonus (9.7 nmol) or forelimb tonic extension (11.7 nmol) 30 min after injection
of tested compounds. c Significant differences between ED50 values of the group treated with aniracetam + 2,3-benzodiazepine and the
d
group treated with 2,3-benzodiazepine alone (Table 1) are denoted: *p < 0.01. Ref 15.
internal standard and coupling constants (J ) in Hz. All
exchangeable protons were confirmed by addition of D2O.
Compounds 2a -d and 3a -d were prepared according to a
procedure previously described;15,16 derivatives 2b and 3b are
new products.
3,5-Dih yd r o-7,8-d im eth oxy-1-(4-flu or op h en yl)-4H-2,3-
1
ben zod ia zep in -4-on e (2b). Mp: 208-211 °C, yield 51%. H
NMR: 3.50 (s, 2H, CH2), 3.73 (s, 3H, MeO-7), 3.97 (s, 3H, MeO-
8), 6.64 (s, 1H, H-6), 6.85 (s, 1H, H-9), 7.12 (dd, 2H, J HH ) 8.5
and J HF ) 8.7, H-3′,5′), 7.63 (dd, 2H, J HF ) 5.4 and J HH ) 8.5,
H-2′,6′), 8.45 (bs, 1H, NH). Anal. (C17H15FN2O3) C, H, N.
3,5-Dih yd r o-7,8-d im eth oxy-1-(4-flu or op h en yl)-4H-2,3-
ben zod ia zep in e-4-th ion e (3b). Mp: 176-179 °C, yield 89%.
1H NMR: 3.73 (s, 3H, MeO-7), 3.95 (bs, 2H, CH2), 3.97 (s, 3H,
MeO-8), 6.60 (s, 1H, H-6), 6.87 (s, 1H, H-9), 7.14 (dd, 2H, J HH
) 8.5 and J HF ) 8.8, H-3′,5′), 7.63 (dd, 2H, J HF ) 5.5 and J HH
) 8.5, H-2′,6′), 9.93 (bs, 1H, NH). Anal. (C17H15FN2O2S) C, H,
N.
F igu r e 1. Time profiles of plasma levels after ip administra-
tion of 6f in rats. The ordinate shows the plasma level; the
abscissa shows the time after ip administration of drug in
minutes.
Gen er a l P r oced u r e for Syn th esis of 11H-[1,2,4]Tr ia -
zolo[4,5-c][2,3]ben zod ia zep in -3(2H)-on es 6a -d . A solution
of 2,3-benzodiazepine-4-thione 3a -d (1 mmol) and ethyl
carbazate (1.5 mmol) in n-BuOH (20 mL) was refluxed for 24-
30 h. The solution was evaporated to dryness, the oil crystal-
lized by adding a small amount of acetone, and the products
6a -d were recrystallized from EtOAc.
8,9-Dim eth oxy-6-p h en yl-11H-[1,2,4]tr ia zolo[4,5-c][2,3]-
ben zod ia zep in -3(2H)-on e (6a ). Mp: >300 °C, yield 62%. 1H
NMR: 3.68 (s, 3H, MeO-8), 3.84 (s, 2H, CH2), 3.97 (s, 3H, MeO-
9), 6.65 (s, 1H, H-7), 6.85 (s, 1H, H-10), 7.41-7.81 (m, 5H,
ArH), 9.58 (bs, 1H, NH). Anal. (C18H16N4O3) C, H, N.
8,9-Dim eth oxy-6-(4-flu or op h en yl)-11H-[1,2,4]tr ia zolo-
[4,5-c][2,3]ben zod ia zep in -3(2H)-on e (6b). Mp: >300 °C,
yield 64%. 1H NMR: 3.69 (s, 3H, MeO-8), 3.83 (s, 2H, CH2),
3.97 (s, 3H, MeO-9), 6.61 (s, 1H, H-7), 6.85 (s, 1H, H-10), 7.13
F igu r e 2. Anticonvulsant effects of 2f, 6f, and 1 (33 µmol/
kg) against audiogenic seizures in DBA/2 mice. The ordinate
shows seizure score; the abscissa shows the time after ip
administration of drug in minutes. Ten animals were used for
the determination of each point.
(dd, 2H, J HH ) 8.5 and J HF ) 8.8, H-3′,5′), 7.82 (dd, 2H, J HF
)
5.3 and J HH ) 8.5, H-2′,6′), 9.77 (bs, 1H, NH). Anal. (C18H15
-
FN4O3) C, H, N.
8,9-Dim et h oxy-6-(3-n it r op h en yl)-11H -[1,2,4]t r ia zolo-
[4,5-c][2,3]ben zod ia zep in -3(2H)-on e (6c). Mp: >300 °C,
yield 58%. 1H NMR: 3.69 (s, 3H, MeO-8), 3.81 (s, 2H, CH2),
4.00 (s, 3H, MeO-9), 6.56 (s, 1H, H-7), 6.99 (s, 1H, H-10), 7.70
(t, 1H, J ) 8.0, H-5′), 8.38 (m, 2H, H-4′,6′), 8.49 (d, 2H, J )
1.8, H-2′), 8.80 (bs, 1H, NH). Anal. (C18H15N5O5) C, H, N.
8,9-Dim et h oxy-6-(4-n it r op h en yl)-11H -[1,2,4]t r ia zolo-
[4,5-c][2,3]ben zod ia zep in -3(2H)-on e (6d ). Mp: >300 °C,
yield 54%. 1H NMR: 3.69 (s, 3H, MeO-8), 3.87 (s, 2H, CH2),
3.99 (s, 3H, MeO-9), 6.53 (s, 1H, H-7), 6.87 (s, 1H, H-10), 8.07
(d, 2H, J ) 8.9, H-2′,6′), 8.30 (d, 2H, J ) 8.9, H-3′,5′), 8.65 (bs,
1H, NH). Anal. (C18H15N5O5) C, H, N.
time of action than 1 and 2. They undergo biotransfor-
mation into the corresponding bicyclic derivatives 2
which are proposed to be the main agents responsible
for the anticonvulsant activity of the tricyclic derivatives
in various seizure models. Also in this series of AMPA
antagonists the trend that the 4-aminophenyl derivative
is more potent than other analogues has been confirmed.
Gen er a l P r oced u r e for Syn th esis of Am in op h en yl
Der iva tives 6e,f. A mixture of nitro derivative 6c or 6d (0.2
mmol) and SnCl2‚2H2O (1 mmol) in EtOH (20 mL) was heated
on a boiling water bath for 90 min. The mixture was cooled,
poured in water, neutralized with a solution of NaHCO3 and
extracted with EtOAc. The organic phase was dried over Na2-
SO4, the solvent was evaporated and from the crude residue
compounds 6e,f were purified by column chromatography
using CHCl3/MeOH (90:10) as eluant.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Kofler
hot stage apparatus and are uncorrected. Elemental analyses
(C, H, N) were carried out on a Carlo Erba model 1106
elemental analyzer and the results are within 0.4% of the
theoretical values. Merck silica gel 60 F254 plates were used
for analytical TLC; column chromatography was performed on
Merck silica gel 60 (70-230 mesh). 1H NMR spectra were
measured in CDCl3 with a Varian Gemini 300 spectrometer;
chemical shifts are expressed in δ (ppm) relative to TMS as
6-(3-Am in op h en yl)-8,9-d im eth oxy-11H-[1,2,4]tr ia zolo-
[4,5-c][2,3]ben zod ia zep in -3(2H)-on e (6e). Mp: >300 °C,

4838 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Zappala` et al.
yield 65.3%. 1H NMR: 3.70 (s, 3H, MeO-8), 3.80 (bs, 2H, NH2),
3.81 (s, 2H, CH2), 3.96 (s, 3H, MeO-9), 6.69 (s, 1H, H-7), 6.80
(m, 1H, H-4′), 6.82 (s, 1H, H-10), 6.89 (m, 1H, H-6′), 7.17 (t,
1H, J ) 7.8, H-5′), 7.34 (m, 2H, H-2′). Anal. (C18H17N5O3) C,
H, N.
for clonus was 1.76 nmol (1.06-3.07), while that for tonus was
2.90 (1.83-4.58) nmol. For icv injection, the mice were
anesthetized with diethyl ether, and injections were made in
the left or right lateral ventricle (coordinates 1 mm posterior
and 1 mm lateral to the bregma; depth 2.4 mm) using a 10-µL
Hamilton microsyringe (type 701N) fitted with a nylon cuff
on the needle as previously described.27 Injections of drugs by
this procedure led to a uniform distribution throughout the
ventricular system within 10 min. The animals were placed
singly in a 30- × 30- × 30-cm box, and the observation time
was 30 min after the administration of AMPA.
6-(4-Am in op h en yl)-8,9-d im eth oxy-11H-[1,2,4]tr ia zolo-
[4,5-c][2,3]ben zod ia zep in -3(2H)-on e (6f). Mp: >300 °C,
1
yield 40.5%. H NMR: 3.72 (s, 3H, MeO-8), 3.79 (s, 2H, CH2),
3.97 (s, 3H, MeO-9), 4.01(bs, 2H, NH2), 6.74 (s, 1H, H-7), 6.69
(d, 2H, J ) 8.6, H-3′,5′), 6.82 (s, 1H, H-10), 7.64 (d, 2H, J )
8.6, H-2′,6′), 8.41 (bs, 1H, NH). Anal. (C18H17N5O3) C, H, N.
Lip op h ilicity Mea su r em en ts. The relative lipophilicity
(Rm) of the compounds was measured by reversed-phase high-
performance thin-layer chromatography (RP-HPTLC) accord-
ing to the method previously described.22 Briefly, Whatman
KC18F plates were used as the nonpolar stationary phase. The
plates were dried at 105 °C for 1 h before use. The polar mobile
phase was a 2:1 (v/v) mixture of acetone and water. Each
compound was dissolved in CHCl3 (3 mg/mL), and 1 µL of
solution was applied onto the plate. The experiments were
repeated five times with a different disposition of the com-
pounds on the plate. The Rf values were expressed as the mean
values of the five determinations. The Rm values were calcu-
lated from the experimental Rf values according to the
formula: Rm ) log[(1/Rf) - 1]. Higher Rm values indicate
higher lipophilicity.
Testin g of An ticon vu lsa n t Activity. 1. Au d iogen ic
Seizu r es in DBA/2 Mice. All experiments were performed
with DBA/2 mice which are genetically susceptible to sound-
induced seizures.23 DBA/2 mice (8-12 g, 22-25 days old) were
purchased from Charles River (Calco, Como, Italy). Groups of
10 mice of either sex were exposed to auditory stimulation 30
min following administration of vehicle or each dose of drugs
studied. The compounds were given ip (0.1 mL/10 g of body
weight of the mouse) as a freshly prepared solution in 50%
dimethyl sulfoxide (DMSO) and 50% sterile saline (0.9% NaCl).
Individual mice were placed under a hemispheric Perspex
dome (diameter 58 cm), and 60 s was allowed for habituation
and assessment of locomotor activity. Auditory stimulation
(12-16 kHz, 109 dB) was applied for 60 s or until tonic
extension occurred and induced a sequential seizure response
in control DBA/2 mice, consisting of an early wild running
phase, followed by generalized myoclonus and tonic flexion and
extension sometimes followed by respiratory arrest. The
control and drug-treated mice were scored for latency to and
incidence of the different phases of the seizures.24 The time
course of the anticonvulsant action of 6f (33 µmol/kg) was
determined following administration to groups of 10 mice that
were tested for sound-induced seizure responses at 5-210 min
after drug administration.
2. MES Test in Sw iss Mice. Male Swiss mice (20-26 g,
42-48 days old) were purchased from Charles River (Calco,
Como, Italy). Electrical stimuli were applied via ear-clip
electrodes to the mice (rectangular constant current impulses,
amplitude 50 mA, width 20 ms, frequency 35 Hz, duration 400
ms) according to the method of Swinyard et al.25 Abolition of
tonic hindlimb extension after drug treatment was considered
as the endpoint of protection. In general, the dose-response
curves were estimated by testing 4-5 doses using 8-10 mice
for each dose.
3. P TZ-In d u ced Seizu r es in Sw iss Mice. Male Swiss
mice were pretreated with vehicle or drug 45 min before the
subcutaneous (sc) administration of pentylenetetrazole. For
systemic injections, all tested compounds were given ip (0.1
mL/10 g of body weight of the mouse) as a freshly prepared
solution in 50% DMSO and 50% sterile saline (0.9% NaCl).
The convulsive dose 97 (CD97) of PTZ (85 mg/kg) was applied
and the animals were observed for 30 min. A threshold
convulsion was an episode of clonic spasms lasting for at least
5 s. The absence of this threshold convulsion over 30 min
indicated that the tested substance had the ability to elevate
PTZ seizure threshold.26
5. P r etr ea tm en t w ith An ir a ceta m . The icv microinjection
of aniracetam was performed in accordance with experimental
procedures previously described for AMPA microinjection.27
The dose of aniracetam (50 nmol icv) was administered 60 min
before auditory stimulation or 30 min before each compound
in DBA/2 mice.
Effects on Motor Movem en ts. Groups of 10 mice were
trained to make coordinated motor movements continuously
for 2 min on a rotarod, 3-cm diameter, at 8 rpm (U. Basile,
Comerio, Varese, Italy). Impairment of coordinated motor
movements was defined as the inability of the mice to remain
on the rotarod for a 2-min test period.28 The ability of the mice
to remain on the rotarod was tested 30 min after administra-
tion of various compounds.
HP LC Assa y. Plasma levels of 6f were quantified by high-
performance liquid chromatography (HPLC) with ultraviolet
(UV) detection, according to a suitable modified method
recently described by Rizzo et al.18 Briefly, the method included
a single-step extraction and the use of internal standard for
quantitation. The HPLC system consisted of a J asco LG-980-
02 ternary gradient unit with a J asco PU-980 pump and a 20-
µL loop injection valve; the detector used was a J asco UV-975
set at 240 nm; all data were elaborated by a Borwin chroma-
tography software, version 1.21. A Hypersil ODS 5-µm (250-
× 4.6-mm) column with an ODS guard column was used. The
column was eluted isocratically at room temperature with
phosphate buffer/MeOH/MeCN (57:17:26, v/v), pH 5.8, at a
flow rate of 1 mL/min. Stock solution (1 mg/mL) of 6f was
prepared in MeOH and refrigerated at 4 °C. Working solutions
were made by appropriate dilution in MeOH and used to
prepare plasma standards for the calibration curves. For the
extraction procedures an aliquot of 0.5 mL of rat plasma was
mixed with 0.5 mL of carbonate buffer, pH 9.0. The sample
was applied to Extrelut NT 1 (Merck, Germany), a prepacked
glass column. After 10 min, Et2O (6 mL) was added to the
column. The eluate was collected and evaporated to dryness
under a stream of nitrogen. The residue was dissolved in 100
µL of mobile phase and a 20-µL aliquot was injected into the
chromatographic system. A linear response was observed over
the examined concentration range (0.5-5 µg/mL). The lower
limit of detection was 70 ng/mL for 6f.
St a t ist ica l An a lysis. Statistical comparisons between
groups of control and drug-treated animals were made using
Fisher’s exact probability test (incidence of the seizure phases).
The ED50 value of each phase of the audiogenic seizure or
seizures induced by MES or PTZ was determined for each dose
of compound administered, and dose-response curves were
fitted using a computer program by the Litchfield and Wil-
coxon’s method.29 The relative anticonvulsant activities were
determined by comparison of respective ED50 values. Statisti-
cal significance between control and test groups of data means
was tested using a two-tailed Student’s t-test.
Ack n ow led gm en t. Financial support for this re-
search by Fondo Ateneo di Ricerca (1999, Messina, Italy)
and CNR (#99.00987.CT03, Rome, Italy) is gratefully
acknowledged.
Refer en ces
4. AMP A-In d u ced Seizu r es in DBA/2 Mice. Seizures
(1) Doble, A. Excitatory Amino Acid Receptors and Neurodegen-
were also induced by icv injection of AMPA. The CD50 of AMPA
eration Therapie 1995, 50, 319-337.

11H-[1,2,4]Triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4839
(2) (a) Muir, K. W.; Lees, K. R. Clinical Experience with Excitatory
Amino Acid Antagonist Drugs. Stroke 1995, 26, 503-513. (b)
McEntee, W. C.; Crook, T. H. Glutamate, its Role in Learning,
Memory, and the Aging Brain. Psychopharmacology 1993, 111,
391-401.
(3) (a) Dingledine, R.; Borges, K., Bowie, D.; Traynelis, S. R. The
Glutamate Receptor Ion Channels. Pharmacol. Rev. 1999, 51,
7-61. (b) Monaghan, D. T., Wenthold, R. J ., Eds. The Ionotropic
Glutamate Receptors; Humana Press: Totowa, NJ , 1997.
(4) Rosenmund, C.; Stern-Bach, Y.; Stevens, C. F. The Tetrameric
Structure of a Glutamate Receptor Channel. Science 1998, 280,
1596-1599.
(5) Sutcliffe, M. J .; Wo, Z. G.; Oswald, R. E. Three-Dimensional
Models of non-NMDA Glutamate Receptors. Biophys. J . 1996,
70, 1575-1589.
(16) Chimirri, A.; De Sarro, G.; De Sarro, A.; Gitto, R.; Quartarone,
S.; Zappala`, M.; Constanti, A.; Libri, V. 3,5-Dihydro-4H-2,3-
benzodiazepine-4-thiones:
a New Class of AMPA Receptor
Antagonists. J . Med. Chem. 1998, 41, 3409-3416.
(17) (a) De Sarro, G.; Rizzo, M.; Sinopoli, V. A.; Gitto, R.; De Sarro,
A.; Zappala`, M.; Chimirri, A. Relationship between Anticonvul-
sant Activity and Plasma Level of some 2,3-Benzodiazepines in
Genetically Epilepsy Prone Rats. Pharmacol. Biochem. Behav.
1998, 61, 215-220. (b) De Sarro, G.; Rizzo, M.; Spagnolo, C.;
Gitto, R.; De Sarro, A.; Scotto, G., Zappala`, M.; Chimirri, A.,
Anticonvulsant Activity and Plasma Level of Some 2,3-Benzo-
diazepin-4-ones (CFMs) in Genetically Epilepsy Prone Rats.
Pharmacol. Biochem. Behav. 1999, 63, 621-627.
(18) (a) Rizzo, M.; Sinopoli, V. A.; Gitto, R.; Zappala`, M.; De Sarro,
G.; Chimirri, A. High-Performance Liquid Chromatography
Determination of New 2,3-Benzodiazepines. J . Chromatogr. B
1998, 705, 149-153. (b) Rizzo, M.; De Sarro, G.; Zappala`, M.;
Chimirri, A. Determination of New 2,3-Benzodiazepines in Rat
Plasma Using High-Performance Liquid Chromatography with
Ultraviolet Detection. J . Chromatogr. B 1999, 731, 207-215.
(19) (a) Chimirri, A.; Zappala`, M.; Gitto, R.; Quartarone, S.; Bevacqua,
F. Synthesis and Structural Features of 11H-tetrazolo[1,5-c][2,3]-
benzodiazepines. Heterocycles 1999, 51, 1303-1309. (b) Chimirri,
A.; Bevacqua, F.; Gitto, R.; Quartarone, S.; Zappala`, M.; De
Sarro, A.; Maciocco, L.; Biggio, G.; De Sarro, G. Synthesis and
Anticonvulsant Activity of New 11H-triazolo[4,5-c][2,3]benzo-
diazepines. Med. Chem. Res. 1999, 9, 203-212.
(20) (a) Chapman, A. G.; Croucher, M. J .; Meldrum, B. S. Evaluation
of Anticonvulsant Drugs in DBA/2 Mice with Sound-Induced
Seizures. Arzneim. Forsch. 1984, 34, 1261-1264. (b) Engstrom,
F. L.; Woodbury, D. M. Seizure Susceptibility in DBA/2 and C57
Mice: the Effects of Various Convulsants. Epilepsia 1988, 29,
389-395.
(21) Ito, I.; Tanabe, S.; Kohda, A.; Sugiyama, H. Allosteric Potentia-
tion of Quisqualate Receptors by Nootropic Drug Aniracetam.
J . Physiol. (London) 1990, 424, 533-543.
(22) Pietrogrande, M. C.; Borea, P. A.; Biagi, G. L. Lipophilicity
Measurements of Benzodiazepine-Receptor Ligands by Reversed-
Phase Liquid Chromatography. Comparison between High-
Performance Liquid and Thin-Layer Chromatography. J . Chro-
matogr. 1988, 447, 404-409.
(23) Collins, R. L. Audiogenic Seizures. In Experimental Models of
Epilepsy; Purpura, P., Penry, J . K., Tower, D., Woodbury, D. M.,
Walter, R., Eds.; Raven Press: New York, 1972; pp 347-372.
(24) De Sarro, G. B.; Croucher, M. J .; Meldrum, B. S. Anticonvulsant
Actions of DS 103-282: Pharmacological Studies in Rodents and
the Baboon Papio papio. Neuropharmacology 1984, 23, 526-
530.
(25) Swinyard, E. A.; Brown, W. C.; Goodman, L. S. Comparative
Assays of Antiepileptic in Mice and Rats. J . Pharmacol. Exp.
Ther. 1952, 106, 319-330.
(26) Swinyard, E. A.; Woodhead, J . M. Experimental Detection,
Quantification and Evaluation of Anticonvulsants. In Epileptic
Drugs; Woodbury, D. M., Penry, J . K., Pippenger, C. E., Eds.;
Raven Press: New York, 1982; pp 111-126.
(27) De Sarro, G. B.; Ongini, E.; Bertorelli, R.; Aguglia, U.; De Sarro,
A. Excitatory Amino Acid Neurotransmission through both
NMDA and Non-NMDA Receptors is Involved in the Anticon-
vulsant Activity of Felbamate in DBA/2 mice. Eur. J . Pharmacol.
1994, 262, 11-19.
(6) (a) Gill, R.; Lodge, D. Pharmacology of AMPA Antagonists and
their Role in Neuroprotection. Neuroprotective Agents and
Cerebral Ischaemia; Academic Press: San Diego, CA, 1997; pp
197-232. (b) Lees, G. J . Therapeutic Potential of AMPA/Kainate
Receptor Ligands and their Therapeutic Potential in Neurologi-
cal Disorders. Drugs 2000, 59, 33-78.
(7) (a) Lin, Z.; Kadaba, P. K. Molecular Targets for the Rational
Design of Antiepileptic Drugs and Related Neuroprotective
Agents. Med. Res. Rev. 1997, 17, 537-572. (b) Rogawski, M. A.;
Donevan, S. D. AMPA Receptors in Epilepsy and as Targets for
Antiepileptic Drugs. Adv. Neurol. 1999, 79, 947-963.
(8) Chimirri, A.; Gitto, Zappala`, M. AMPA Receptor Antagonists.
Exp. Opin. Ther. Patents 1999, 9, 557-570.
(9) Donevan, S. D.; Rogawski, M. A. GYKI 52466, a 2,3-Benzodi-
azepine, is a Highly Selective, Noncompetitive Antagonist of
AMPA/Kainate Receptor Responses. Neuron 1993, 10, 51-59.
(10) (a) Rogawski, M. A. Therapeutic Potential of Excitatory Amino
Acid Antagonists: Channel Blockers and 2,3-Benzodiazepines.
Trends Pharmacol. Sci. 1993, 14, 325-331. (b) Donevan, S. D.;
Yamaguchi, S. I.; Rogawski, M. A. Non-N-methyl-D-aspartate
Receptor Antagonism by 3-N-Substituted 2,3-Benzodiazepines:
Relationship to Anticonvulsant Activity. J . Pharmacol. Exp.
Ther. 1994, 271, 25-29.
(11) (a) Lodge, D.; Bond, A.; O’Neill, M.; Hicks, C. A.; J ones, M. G.
Stereoselective Effects of 2,3-Benzodiazepines In Vivo: Electro-
physiology and Neuroprotection Studies. Neuropharmacology
1996, 35, 1681-1688. (b) Bleakman, D.; Ballyk, B. A.; Schoepp,
D. D.; Palmer, A. J .; Bath, C. P.; Sharpe, E. F.; Wooley, M. L.;
Bufton, H. R.; Kamboj, R. K.; Tarnawa, I.; Lodge, D. Activity of
2,3-Benzodiazepines at Native Rat and Recombinant Human
Glutamate Receptors In Vitro: Stereospecificity and Selectivity
Profiles. Neuropharmacology 1996, 35, 1689-1702.
(12) (a) Anderson, B. A.; Harn, N. K.; Hansen, M. M.; Harkness A.
R.; Lodge, D.; Leander J . D. Synthesis and Anticonvulsant
Activity of 3-Aryl-5H-2,3-benzodiazepine AMPA Antagonists.
Bioorg. Med. Chem. Lett. 1999, 9, 1953-1956. (b) Wang, Y.;
Konkoy, C. S.; Ilvy, V. I.; Vanover, K. E.; Carter, R.; Weber, E.;
Keana, J . F. W.; Woodward, R. M.; Cai, S. X. Synthesis of 7,8-
(Methylenedioxy)-1-phenyl-3,5-dihydro-4H-2,3-benzodiazepin-4-
ones as Novel and Potent Noncompetitive AMPA Receptor
Antagonists. J . Med. Chem. 1998, 41, 2621-2625.
(13) (a) De Sarro, G.; De Sarro, A.; Gitto, R.; Grasso, S.; Micale, N.;
Quartarone, S.; Zappala`, M. 7,8-Methylenedioxy-4H-2,3-benzo-
diazepin-4-ones as Novel AMPA Receptor Antagonists. Bioorg.
Med. Chem. Lett., 1998, 8, 971-976. (b) De Sarro, G.; De Sarro,
A.; Gitto, R.; Grasso, S.; Micale, N.; Zappala`, M. Synthesis and
Anticonvulsant Activity of New 2,3-Benzodiazepines as AMPA
Receptor Antagonists. Farmaco 1999, 54, 178-187. (c) Grasso,
S.; De Sarro, G.; De Sarro, A.; Micale, N.; Zappala`, M. Puja, G.;
Baraldi, M.; De Micheli, C. Synthesis and Anticonvulsant
Activity of Novel and Potent 2,3-Benzodiazepine AMPA/Kainate
Receptor Antagonists. J . Med. Chem. 1999, 42, 4414-4421.
(14) De Sarro, G.; Chimirri, A.; De Sarro, A.; Gitto, R.; Grasso, S.;
Giusti, P.; Chapman, A. G. GYKI 52466 and Related 2,3-
Benzodiazepines as Anticonvulsant Agents in DBA/2 Mice. Eur.
J . Pharmacol. 1995, 294, 411-422.
(28) Dunham, N. W.; Miya, T. S. A Note on a Simple Apparatus for
Detecting Neurological Deficit in Rats and Mice. J . Am. Pharm.
Assoc. 1957, 46, 208-209.
(29) Litchfield, J . T.; Wilcoxon, F. A Simplified Method of Evaluating
Dose-Effects Experiments. J . Pharmacol. Exp. Ther. 1949, 96,
99-113.
(15) Chimirri, A.; De Sarro, G.; De Sarro, A.; Gitto, R.; Grasso, S.;
Quartarone, S.; Zappala`, M.; Giusti, P.; Libri, V.; Constanti, A.;
Chapman, A. G. 1-Aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-
ones: Novel AMPA Receptor Antagonists. J . Med. Chem. 1997,
40, 1258-1269.
J M001012Y