Synthesis and evaluation of pharmacological properties of novel annelated 2,3-benzodiazepine derivatives

Article abstract of DOI:10.1021/jm030821p

New cyclofunctionalized 2,3-benzodiazepines characterized by a triazolone or triazindione ring fused on the "c" edge of the heptatomic nucleus have been prepared. These compounds were evaluated as potential anticonvulsant agents, and some of them proved t

Full text of DOI:10.1021/jm030821p

3758
J . Med. Chem. 2003, 46, 3758-3761
Syn th esis a n d Eva lu a tion of P h a r m a cologica l P r op er ties of Novel An n ela ted
2,3-Ben zod ia zep in e Der iva tives
Rosaria Gitto,*,# Vale´rie Orlando,# Silvana Quartarone,# Giovambattista De Sarro,§ Angela De Sarro,†
Emilio Russo,§ Guido Ferreri,§ and Alba Chimirri#
Dipartimento Farmaco-Chimico, Universita` di Messina, Viale Annunziata, 98168 Messina, Italy, Dipartimento di Medicina
Sperimentale e Clinica, Universita` degli Studi Magna Græcia di Catanzaro, Catanzaro, Italy, and Istituto di Farmacologia,
Facolta` di Medicina e Chirurgia, Universita` di Messina, Messina, Italy
Received February 21, 2003
New cyclofunctionalized 2,3-benzodiazepines characterized by a triazolone or triazindione ring
fused on the “c” edge of the heptatomic nucleus have been prepared. These compounds were
evaluated as potential anticonvulsant agents, and some of them proved to be more potent
noncompetitive 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) receptor an-
tagonists. In particular, 8,9-dimethoxy-6-(4-bromophenyl)-11H-[1,2,4]triazolo[4,5-c][2,3]benzo-
diazepin-3(2H)-one (5b) was almost 10-fold more active than GYKI-52466 and 3.5-fold more
active than Talampanel. Furthermore, 5b potently reduced AMPA-evoked currents in elec-
trophysiological experiments.
Ch a r t 1. 2,3-Benzodiazepine Derivatives
In tr od u ction
Excessive activation of the ionotropic glutamate re-
ceptors (iGluRs) may be involved in acute and chronic
neurological diseases and in particular in the etiology
of many forms of epilepsy. iGluRs are subdivided into
three primary families based on the agonist that pref-
erentially activates the receptor: N-methyl-D-aspartate
(NMDA), kainate (KA), and 2-amino-3-(3-hydroxy-5-
methylisoxazol-4-yl)propionate (AMPA).1-3
A selective noncompetitive blockade of the AMPA
receptor (AMPAR) was shown by some 2,3-benzodi-
azepines,4,5 such as the 1-(4-aminophenyl)-4-methyl-7,8-
methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466)
(Chart 1) and in particular the 3-N-acetyl-1-(4-ami-
nophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-2,3-
benzodiazepine (2, Talampanel), which aroused great
interest as an anticonvulsant agent and whose phase
II/III clinical trials are underway.6
In our search for new AMPAR negative modulators,7-14
we dentified 1-aryl-3,5-dihydro-7,8-dimethoxy-4H-2,3-
benzodiazepin-4-ones (3, CFM), and thiocarbonyl ana-
logues 4 (Chart 1) showing anticonvulsant effects me-
diated through the AMPAR in a selective and non-
competitive fashion.7-8
To optimize the potency of our initial lead structures
and provide tools through which to define the require-
ments for negative allosteric AMPAR ligands, we ob-
tained a variety of related compounds such as phthala-
zines9 and annelated 2,3-benzodiazepines.10,11 SAR
studies revealed that an important structural require-
ment necessary to retain anticonvulsant activity con-
sisted of the presence of suitable chemical functionalities
able to engage in hydrogen bonding and that the
pharmacological profile was deeply influenced by the
substituents on the phenyl moiety and the nature of the
fused ring on the heptatomic nucleus. In particular, the
11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-one de-
rivatives (5) showed interesting properties and allowed
us to clarify some SAR aspects.10
In an attempt to further examine the role of cyclo-
functionalization and to gain more information from
SAR studies, we report in this paper the synthesis and
pharmacological studies of other triazolo-2,3-benzodi-
azepines
5 and 2,12-dihydro-9,10-dimethoxy[1,2,4]-
triazino[4,3-c][2,3]benzodiazepine-3,4-diones 7 with a
six-membered ring fused to the heptatomic skeleton.
Ch em istr y
The cyclofunctionalized 2,3-benzodiazepines 5 and 7
were prepared through a multistep synthetic pathway
(Scheme 1) using as intermediates compounds 3, 4, and
* To whom correspondence should be addressed. Phone: +39 090
6766413. Fax: +39 090 355613. E-mail: gittoros@pharma.unime.it.
#
Dipartimento Farmaco-Chimico, Universita` di Messina.
6
obtained according to our previously reported
§ Universita` degli Studi Magna Græcia di Catanzaro.
† Istituto di Farmacologia, Universita` di Messina.
strategies.7-9 In particular, 3,5-dihydro-4H-2,3-benzo-
10.1021/jm030821p CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/10/2003

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3759
Sch em e 1a
Ta ble 1. Anticonvulsant Activity of 2,3-Benzodiazepine
Derivatives against Audiogenic Seizures in DBA/2 Mice
a
ED50
clonic phase
,
µmol/kg
tonic phase
compd
1b
35.8 (24.4-52.4)
13.4 (10.1-17.8)
15.0 (9.01-24.0)
3.65 (2.51-5.31)
28.8 (18.3-45.3)
16.1 (11.8-22.0)
33.6 (22.6-49.9)
76.6 (60.8-96.7)
37.4 (24.6-57.0)
39.2 (24.9-61.7)
>120
25.3 (16.0-40.0)
9.70 (7.00-13.4)
12.6 (8.01-19.0)
1.34 (1.01-1.78)
14.1 (9.17-21.8)
10.2 (8.67-11.9)
17.9 (11.8-27.1)
54.6 (41.5-71.8)
33.0 (24.0-45.4)
28.0 (15.3-51.1)
88.9 (69.2-114)
25.7 (15.9-41.6)
57.7 (45.7-76.1)
52.8 (40.6-68.8)
2b
3h b
5b
5c
5h c
7a
7b
7c
7d
7e
7f
60.0 (45.1-79.9)
74.5 (60.1-92.4)
59.3 (44.5-78.9)
7g
7h
a
All data were calculated according to the method of Litchfield
and Wilcoxon.16 95% confidence limits are given in parentheses.
Reference 14. c Reference 10.
b
Ta ble 2. ED50 Values against MES and PTZ Induced Seizures
in Swiss Mice
a
ED50
,
µmol/kg
compd
MES
PTZ
1b
35.7 (29.3-43.4)
28.8 (23.5-35.3)
15.9 (7.30-33.5)
5.93 (3.54-9.93)
NDd
36.6 (21.6-62.1)
37.8 (27.6-51.8)
68.3 (56.2-83.1)
56.3 (34.2-92.7)
22.6 (11.7-43.8)
13.8 (8.54-22.4)
33.1 (17.5-62.5)
57.6 (34.3-96.7)
63.2 (45.6-87.6)
2c
3h b
5b
5c
7a
7d
a
Reagents: (i) Lawesson’s reagent, dry toluene, 4, 90-120 min;
(ii) H2NNHCO2Et, n-BuOH, 4, 24 h; (iii) N2H4‚H2O, THF, room
temp, 60 min; (iv) Cl2C2O2, dry toluene, room temp, 120 min; (v)
H2/Raney Ni, MeOH, room temp, 60 min.
a
All data were calculated according to the method of Litchfield
and Wilcoxon.16 95% confidence limits are given in parentheses.
Reference 8. c Reference 14 ND ) not determined.
b
d
diazepin-4-ones 3 were activated by transformation into
the corresponding thiocarbonyl derivatives 4 and suc-
cessively converted either into 11H-[1,2,4]triazolo[4,5-
c][2,3]benzodiazepin-3(2H)-one derivatives 5 or 3,5-
dihydro-4H-2,3-benzodiazepine-4-hydrazinyl derivatives
6. By reflux of hydrazinyl derivatives 6 with oxalyl
chloride, a series of new 2,12-dihydro-9,10-dimethoxy-
[1,2,4]triazino[4,3-c][2,3]benzodiazepine-3,4-diones 7 was
synthesized. Aminophenyl-substituted derivatives 7g
and 7h were prepared by reduction of the corresponding
nitro analogues 7e and 7f, respectively. The structures
of the compounds obtained were supported by elemental
analyses and spectroscopic measurements (1H NMR).
dimethoxy[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-
one (5b) is almost 10-fold more active than GYKI 52466
and 3.5-fold more active than CFM-2 and Talampanel.
Furthermore, it is noteworthy to observe that the
typical aminophenyl substituent that characterizes
some active 2,3-benzodiazepines (e.g., 1, 2, 3h ) is not
always the best for the activity but other functionalities,
such as electron-withdrawing substituents on the phen-
yl moiety, might be useful for optimizing the pharma-
cological properties. This possibility had also been
suggested by our pharmacophore model of noncompeti-
tive AMPAR, in which one of the main structural
features consists of an aromatic ring without taking the
phenyl substituent into consideration.13
Resu lts a n d Discu ssion
The anticonvulsant efficacy of the novel 2,3-benzodi-
azepines 5b-c and 7a -h was evaluated, 30 min after
intraperitoneal (ip) administration, against audiogenic
seizures in DBA/2 mice (Table 1), which has been
considered an excellent animal model for generalized
epilepsy and for screening new anticonvulsant drugs.
The results were compared with those of GYKI 52466
(1), Talampanel (2), CFM-2 (3h ), and 6-(4-aminophenyl)-
11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-one
(5h ) already reported.10 Compounds 7 are generally less
potent than the corresponding 5 and possess an anti-
convulsant potency that is influenced by the substituent
on the phenyl ring. Anticonvulsant screening of triazolo-
2,3-benzodiazepines 5b and 5c indicated that the in-
troduction of a halogen atom on the phenyl ring led to
potent anticonvulsant agents and in particular that the
newly synthesized compound 6-(4-bromophenyl)-8,9-
The most active compounds of series 5 and 7 were
further evaluated against seizures induced by MES and
PTZ in Swiss mice (Table 2) and compared with 2,3-
benzodiazepines 1, 2, and 3h . As shown in Table 2, MES
and PTZ induced seizures were significantly reduced 45
min after ip administration of the studied compounds,
with the same rank order observed in the audiogenic
seizures test, and 5b retained anticonvulsant effects
higher than those of GYKI 52466 (1), Talampanel (2),
and CFM-2 (3h ).
The anticonvulsant properties of 5b and 5c were also
evaluated after pretreatment with aniracetam, a posi-
tive allosteric modulator of AMPAR, reversing the
anticonvulsant properties of some 2,3-benzodiazepines.15
As expected, the icv injection of aniracetam (50 nmol)
60 min before testing reduced the efficacy of 5b and 5c
(Table 3), indicating an involvement of AMPA-mediated

3760 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Brief Articles
Ta ble 3. ED50 Values against Audiogenic Seizures after
Pretreatment with Aniracetam in DBA/2 Mice
a
ED50
,
µmol/kg
pretreatment with aniracetam
compd
clonic phase
tonic phase
1b
134 (88.8-203)
65.4 (44.5-96.2)
18.6 (10.1-34.2)
87.8 (63.4-121)
100 (63.4-158)
56.2 (43.4-77.9)
7.87 (4.61-13.4)
71.8 (53.2-96.9)
3h b
5b
5c
a
All data were calculated according to the method of Litchfield
and Wilcoxon.16 95% confidence limits are given in parentheses.
Reference 8.
b
Ta ble 4. ED50 Values against AMPA-Induced Seizures in
DBA/2 Mice
a
ED50
,
µmol/kg
AMPA-induced seizures
compd
clonic phase
tonic phase
1b
57.5 (43.5-76.0)
44.2 (34.6-56.5)
32.1 (23.2-44.3)
17.7 (13.6-23.0)
46.8 (38.2-57.3)
40.5 (26.3-60.8)
29.1 (22.6-37.5)
25.0 (16.5-30.0)
13.4 (7.10-25.0)
39.9 (18.6-85.5)
2c
3h b
5b
5c
a
All data were calculated according to the method of Litchfield
and Wilcoxon.16 95% confidence limits are given in parentheses.
Reference 8. c Reference 14.
b
neurotransmission in the anticonvulsant properties. As
shown in Table 4, the ip administration of 5b or 5c was
also able to protect against AMPA-induced seizures.
Finally, electrophysiological studies were carried out
on 5b, the most active compound of the series, measur-
ing AMPA receptor-mediated currents (Figure 1) under
voltage clamp in Sprague-Dawley rat olfactory cortical
neurons in vitro (Supporting Information). The results
confirmed that the responses evoked by 0.25-5 µM
AMPA were consistently abolished by 1 µM 5b (Figure
1A), a dose 50-fold lower than those of compounds 1 and
3h .8 The mean peak amplitude of the AMPA-evoked
inward currents was suppressed by ∼7-57% over the
AMPA dose range (Figure 1A). Moreover, from the clear
depression of the apparent maximum of the AMPA
dose-response relation (Figure 1B), it is likely that 5b
was acting via a noncompetitive-type blocking mecha-
nism at the AMPA receptor/ion channel complex.
In conclusion, we have identified new cyclofunction-
alized 2,3-benzodiazepines 5 and 7 that showed anti-
convulsant activity in different seizure models acting
as noncompetitive AMPAR antagonists. In particular,
derivative 5b proved to be in vivo and in vitro more
active than GYKI 52466 (1), CFM-2 (3h ), and Talam-
panel (2). Furthermore, the results herein reported
provide evidence that, in contrast to SAR findings
reported until now, electron acceptor substituents are
useful for optimizing the AMPAR antagonist activity.
F igu r e 1. (A) Chart recordings from a single cortical neuron
clamped at -70 mV (in TTX), showing inward currents evoked
by 0.5, 1, and 2 µM AMPA (1 min bath applications) in control
solution and after 15 min of preincubation with compound 5b
(1 µM). Peak currents were depressed by 52%, 56%, and 57%,
respectively. Scale bars apply to all traces. (B) Noncompetitive-
type depression of AMPA dose-response relation in the
presence of 5b. Peak inward membrane currents induced by
AMPA were measured in rat olfactory cortical brain slice
neurons voltage-clamped at -70 mV in the presence of 1 µM
TTX. Points represent pooled means ((SEM, nA) plotted
against applied AMPA concentration (0.25-5 µM, log scale)
in the absence (O; N ) 7) and presence (b; N ) 7) of 0.5 µM
5b (curves were fitted by eye).
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. 1H NMR spectra were measured with a
Varian Gemini 300 spectrometer. Chemical shifts are ex-
pressed in δ (ppm) relative to TMS as the internal standard,
and coupling constants (J ) are in hertz. All exchangeable
protons were confirmed by addition of D2O.
Compounds 5b and 5c were prepared by refluxing 3,5-
dihydro-4H-2,3-benzodiazepine-4-thiones (4b and 4c) with
ethyl carbazate in n-BuOH as previously reported in our
paper.10
Gen er a l P r oced u r e for th e Syn th esis of 2,12-Dih yd r o-
9,10-d im et h oxy-7-a r yl[1,2,4]t r ia zin o[4,3-c][2,3]b en zod i-
a zep in e-3,4-d ion es (7a -f). To a cooled (5 °C) suspension of

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3761
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compound 6 (0.28 mmol) in toluene (15 mL) was added oxalyl
chloride (3.36 mmol). The mixture was stirred at room tem-
perature for 2 h. The solution was evaporated under vacuum,
and the crude product was crystallized from the suitable
solvent. The starting compounds 6a -f were easily prepared
by treating thiocarbonyl derivatives 4a -f with hydrazine
hydrate according to a procedure previously described.11b
Gen er a l P r oced u r e for th e Syn th esis of Am in op h en yl
Der iva tives (7g a n d 7h ). The nitro derivatives (7e and 7f,
0.36 mmol) were hydrogenated in vacuo by adding Raney Ni
as catalyst to a MeOH solution (30 mL), and the mixture was
stirred at room temperature for 1h. The Raney Ni was filtered
out, and the solvent was removed under vacuum. The resulting
residue was crystallized from CHCl3.
Ack n ow led gm en t. Financial support for this re-
search by Fondo Ateneo di Ricerca (University of Mes-
sina, Italy) and with Grant COFIN2002 is gratefully
acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Spectral data and
experimental procedures for pharmacological evaluation. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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