3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones: Tricyclic heteroaromatic derivatives as a new class of benzodiazepine receptor ligands

Article abstract of DOI:10.1021/jm991131h

A series of 3-substituted [1,2,4]triazino[4,3-c]benzimidazoles V were prepared and tested at the central benzodiazepine receptor (BzR). These compounds were designed as rigid analogues of the previously described N- benzylindolylglyoxylylamide derivatives IV. The title compounds V showed an affinity which depended directly on the presence of the N(10)-H group and an aromatic ring at position 3. Some of them elicited a 2- or 3-fold higher affinity with respect to that of the indolylglyoxylylamide derivatives IV (R=H). The GABA ratio and [³⁵S]-tert-butylcyclophosphorothionate binding data revealed an efficacy profile of partial inverse agonists/antagonists for compounds 1c,e,f,j,k, and of a partial agonist for 2c. This last compound proved to be effective in antagonizing pentylenetetrazole-induced seizures in mice. Attempts were made to interpret the structure-affinity relationships of compounds V in the light of possible tautomeric equilibria involving the ligands.

Full text of DOI:10.1021/jm991131h

96
J . Med. Chem. 2000, 43, 96-102
3-Ar yl-[1,2,4]tr ia zin o[4,3-a ]ben zim id a zol-4(10H)-on es: Tr icyclic Heter oa r om a tic
Der iva tives a s a New Cla ss of Ben zod ia zep in e Recep tor Liga n d s
Giampaolo Primofiore,*,§ Federico Da Settimo,§ Sabrina Taliani,§ Anna Maria Marini,§ Concettina La Motta,§
Ettore Novellino,† Giovanni Greco,† Marco Gesi,# Letizia Trincavelli,‡ and Claudia Martini‡
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica
Farmaceutica e Tossicologica, Universita` di Napoli “Federico II”, Via Domenico Montesano 49, 80131 Napoli, Italy,
Dipartimento di Morfologia Umana e Biologia Applicata, Universita` di Pisa, Via Roma 55, 56126 Pisa, Italy, and
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa,
Via Bonanno 6, 56126 Pisa, Italy
Received August 4, 1999
A series of 3-substituted [1,2,4]triazino[4,3-c]benzimidazoles V were prepared and tested at
the central benzodiazepine receptor (BzR). These compounds were designed as rigid analogues
of the previously described N-benzylindolylglyoxylylamide derivatives IV. The title compounds
V showed an affinity which depended directly on the presence of the N(10)-H group and an
aromatic ring at position 3. Some of them elicited a 2- or 3-fold higher affinity with respect to
that of the indolylglyoxylylamide derivatives IV (R ) H). The GABA ratio and [35S]-tert-
butylcyclophosphorothionate binding data revealed an efficacy profile of partial inverse agonists/
antagonists for compounds 1c,e,f,j,k , and of a partial agonist for 2c. This last compound proved
to be effective in antagonizing pentylenetetrazole-induced seizures in mice. Attempts were made
to interpret the structure-affinity relationships of compounds V in the light of possible
tautomeric equilibria involving the ligands.
In tr od u ction
Benzodiazepine receptor (BzR) ligands allosterically
modulate the action of GABA on neuronal chloride ion
flux, thus eliciting a wide variety of pharmacological
actions ranging in a continuum from full agonists
(anxiolytic, sedative/hypnotic, and anticonvulsant ac-
tivities) to inverse agonists (proconvulsant and anxio-
genic activities), via antagonists, which do not exhibit
per se any pharmacological effects but can antagonize
the action of both agonists and inverse agonists.1-3
To this day, the structure of the BzR remains unre-
solved.4,5 A wide variety of compounds with a chemical
structure different from that of benzodiazepines have
been synthesized and tested6-16 to identify BzR ligands
displaying high potency and the desired efficacy profile.
Among them, the most interesting classes are 2-arylpyra-
zoloquinolines (I),6,10 â-carbolines (II),8 and pyridodiin-
doles (III).11 A comprehensive pharmacophore model for
BzR ligands has recently been proposed by Cook et al.17
which rationalizes the binding affinity and the efficacy
profile of many chemically heterogeneous classes of
compounds in terms of hydrophobic and hydrogen bond
interactions.
derivatives (IV), which were hypothesized to assume a
pseudoplanar arrangement mimicking the â-carboline
system.18 However, compounds IV were generally found
to be less potent than ligands of classes I-III, probably
owing to their greater conformational flexibility.
A useful contribution to the refinement of Cook’s
model came also from some structure-activity relation-
ships (SARs) and molecular modeling studies of a new
class of BzR ligands, the N-benzylindolylglyoxylylamide
* To whom all correspondence should be addressed. Tel: 39 50 50
0209. Fax: 39 50 40 517.
§ Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
† Dipartimento di Chimica Farmaceutica e Tossicologica, Universita`
“Federico II” di Napoli.
#
Dipartimento di Morfologia Umana e Biologia Applicata, Univer-
We recently started a wide research program aimed
at devising new BzR ligands characterized by a higher
degree of rigidity with respect to the N-benzylindolyl
sita` di Pisa.
‡ Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie, Universita` di Pisa.
10.1021/jm991131h CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/10/1999

3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 97
Sch em e 1a
ate glyoxylic acid in ethanol to give the intermediate
substituted (benzimidazol-2-ylhydrazono)acetic acids
5a -c,e-h ,j,m and 6c,e,g,h ,j which were purified by
suspension in hot methanol, as the recrystallization
process always led to partial cyclization of the products
(Scheme 1, Table 1, Supporting Information). The
cyclization of these acids was obtained using one of the
following methods (Scheme 1, Table 2): (A) by heating
the acids at a temperature 30-40 °C above their melting
point for 10 min; (B) by saturating with anhydrous
hydrogen chloride a suspension of the acid in absolute
ethanol at 0 °C, then refluxing the reaction mixture for
4-7 h; (C) by refluxing the acids for 1 h in glacial acetic
acid.
In procedure 2, compounds 3 and 4 were reacted with
the appropriate glyoxylic acid ethyl ester in refluxing
ethanol to give the target compounds 1d ,k ,l and 2d ,k
directly (Scheme 1, Table 2, Supporting Information).
The product 1i was obtained from the corresponding
20
dimethoxy derivative 1h by demethylation with BBr3
in anhydrous dichloromethane (Table 2, Supporting
Information).
Compounds 1c,21d ,g,h were allowed to react with
methyl iodide in absolute ethanol in the presence of
sodium ethylate. Compound 1c gave an approximately
1:2 mixture of 2c and the 1-methylated product 7, while
1d ,g,h gave almost exclusively the 1-methylated prod-
ucts 8-10, isomers of 2d ,g,h , respectively (Scheme 2,
Supporting Information).
a
Reagents: (A) fusion; (B) ethanol, gaseous HCl, reflux; (C)
glacial acetic acid, reflux.
glyoxylylamides IV. The early results of our studies are
reported in the present paper, where we describe the
synthesis and biological evaluation of a series of [1,2,4]-
triazino[4,3-a]benzimidazole derivatives V designed as
geometrically constrained analogues of the indolylgly-
oxylyl system of compounds IV. Figure 1 shows a
superimposition of the molecular models of ligands of
types I, III, and V (R′ ) phenyl), in accordance with
their hypothesized common mode of interaction with the
BzR.
Resu lts a n d Discu ssion
Bin d in g Stu d ies. The binding affinities of the tri-
azinobenzimidazoles 1a -m and 2c-e,j,k at the BzR
were determined in bovine brain membranes by dis-
placement experiments with the radiolabeled antagonist
[3H]Ro 15-1788.22 The in vitro efficacy profile of the most
active compounds was measured by the GABA ratio23-25
and by the [35S]-tert-butylbicyclophosphorothionate ([35S]-
TBPS) binding shift.26-28 The results of the in vitro
assays, listed in Table 3, are here summarized.
Ch em istr y
The triazinobenzimidazole derivatives 1a -m and
2c-e,g,h ,j,k were prepared essentially following two
general procedures. Procedure 1 involved the reaction
of 2-hydrazinobenzimidazole 3 or 419 with the appropri-
Affinity depends directly on the nature of the sub-
stituents at positions 10 and 3. The benzimidazole N(10)
has to be unsubstituted for a high affinity, as its
Sch em e 2

98 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Primofiore et al.
Ta ble 1. Physical Properties of Benzimidazol-2-ylhydrazone
Derivatives 5a -c,e-h ,j,m and 6c,e,g,h ,j
no.
R
R′
yield (%)
mp (°C)
formulaa
5a
5b
5c
5e
5f
5g
5h
5j
H
H
CH3
C6H5
C6H4-4-OCH3
C6H4-4-OH
C6H4-4-CH3
C6H3-3,4-(OCH3)2
fur-2-yl
79
70
78
66
93
87
67
98
66
80
56
73
70
66
186-189
220-222
247-250
278-280
256-259
>300
299-301
>300
228-231
C9H8N4O2
b
H
H
H
H
H
H
H
H
C10H10N4O2
C15H12N4O2
C16H14N4O3
C15H12N4O3
C16H14N4O2
C17H15N4O4
C13H10N4O3
5m
thien-3-yl
C13H10N4O2S
6c CH3 C6H5
211-214 dec C16H14N4O2
6e CH3 C6H4-4-OCH3
6g CH3 C6H4-4-CH3
6h CH3 C6H3-3,4-(OCH3)2
194-197
195-198
210-211
213-215
C17H16N4O3
C17H16N4O2
C18H18N4O4
C14H12N4O3
F igu r e 1. Overlay of the triazinobenzimidazole derivative 1c
of type V (red) on the 4′-chlorophenylpyrazoloquinoline deriva-
tive6 of type I (yellow) and the 2-chloropyridodiindole deriva-
tive30 of type III (cyan). Labels A2, A3, H1, H2, L1, and L2 refer
to BzR subsites in accordance with Cook’s pharmacophore
model.17 Molecular models of the ligands were obtained as
described in the Experimental Section.
6j
CH3 fur-2-yl
a
Elemental analyses for C, H, N were within (0.4% of the
b
calculated values. This product is described in refs 49 and 50
with mp 291 and >360 °C, respectively, very different from the
one reported here. The IR, 1H NMR, and MS spectral data for our
product are fully consistent with the structure assigned (Support-
ing Information).
pendant phenyl ring of 2-arylpyrazoloquinolines I).
Compared with the previously investigated N-benzylin-
dolylglyoxylylamides unsubstituted at the indole 5
position (IV, R ) H),18 these more rigid 3-aryltriazi-
nobenzimidazoles V exhibited a 2- or 3-fold improve-
ment in terms of potency, in agreement with the
rationale leading to their design.
It must be noted that, in compounds of series 1,
potency was highly sensitive to the type of substitution
on the 3-phenyl ring. Insertion of a methyl or a chlorine,
two substituents with opposite electronic effects, at the
4′ position of 1c to give 1g or 1d , respectively, abolished
affinity. These findings cannot be related to an excessive
steric bulk of the 4′-substituent, as 1e,f, which bear a
similarly small-sized group at this position (methoxyl
methylation dramatically lowers affinity (compare series
2 with series 1). The only N(10)-methyl derivative with
an appreciable affinity was 2c, with a Ki value of 860
nM. These results are consistent with the role of a
hydrogen bond donor postulated for the N(10)-H group
at the BzR A2 site (see pharmacophore model in Figure
1).Within series 1, an aromatic ring at position 3
(1c,e,f,i-m ) seemed to be necessary for a high affinity,
since the 3-H- and 3-CH3-substituted compounds 1a ,b,
respectively, were devoid of any affinity. According to
Cook’s model,17 this aryl ring might be involved in a
hydrophobic interaction with the lipophilic site L1 of the
BzR (in Figure 1 the 3-phenyl group of 1c matches the
fused benzene ring E of pyridodiindoles III and the
Ta ble 2. Physical Properties of Triazinobenzimidazole Derivatives 1a -m and 2c-e,g,h ,j,k
no.
R
R′
reaction procedure
yield (%)
recryst solv
mp (°C)
formulaa
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
CH3
C6H5
1C
1C
1A
2
1A
1B
1B
1B
36
30
53
30
39
37
46
61
91
35
43
37
32
37
30
35
36
92
31
19
MeOH
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
EtOH
EtOH
DMF
EtOH
DMF
DMF
DMF
EtOH
DMF
DMF
benzene
288-290
>300
C9H6N4O
H
C10H8N4Ob
C15H10N4Oc
C15H9ClN4O
C16H12N4O2
C15H10N4O2
C16H12N4O
C17H14N4O3
C15H10N4O3
C13H8N4O2
C13H8N4OS
H
>300
H
C6H4-4-Cl
>300
H
C6H4-4-OCH3
C6H4-4-OH
C6H4-4-CH3
C6H3-3,4-(OCH3)2
C6H3-3,4-(OH)2
fur-2-yl
>300
H
>300
H
>300
H
>300
H
>300
1j
1k
1l
H
1B
2
2
298-300
>300
H
thien-2-yl
5-methylthien-2-yl
thien-3-yl
C6H5
C6H4-4-Cl
H
>300
C
C
14H10N4OS
13H8N4OS
1m
2c
2d
2e
2g
2h
2j
H
1B
1A
2
1B
1B
1B
1B
2
>300
CH3
CH3
CH3
CH3
CH3
CH3
CH3
220-221
271-272
241-242
257-259
220-221
294-295
246-248
C16H12N4O
C16H11ClN4O
C17H14N4O2
C17H14N4O
C18H16N4O3
C14H10N4O2
C14H10N4OS
C6H4-4-OCH3
C6H4-4-CH3
C6H3-3,4-(OCH3)2
fur-2-yl
2k
thien-2-yl
a
b
Elemental analyses for C, H, N were within (0.4% of the calculated values. Lit. refs 49 and 50. c Lit. ref 21.

3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 99
Ta ble 3. Inhibition of [3H]Ro 15-1788 Specific Binding to Bovine Brain Membranes by Triazinobenzimidazole Derivatives 1a -l and
2c-e,j,k
% inhibna
(10 µM)
[
35S]TBPS binding with
no.
R
R′
Kib (nM)
NDe
ND
53 ( 2
ND
GABA ratioc
1.20
GABAd (% clonazepam)
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
CH3
C6H5
11
26
97
34
89
99
56
17
99
93
99
83
85
92
30
53
68
63
H
H
35 ( 5
H
C6H4-4-Cl
H
C6H4-4-OCH3
C6H4-4-OH
C6H4-4-CH3
C6H3-3,4-(OCH3)2
C6H3-3,4-(OH)2
fur-2-yl
44 ( 3
74 ( 5
ND
1.25
0.87
5 ( 3
15 ( 5
H
H
H
ND
H
170 ( 8
56 ( 4
13 ( 3
115 ( 11
107 ( 10
860 ( 60
ND
1.20
0.98
0.97
0.96
1.20
1.70
1j
1k
1l
H
25 ( 4
28 ( 3
H
thien-2-yl
5-methylthien-2-yl
thien-3-yl
C6H5
C6H4-4-Cl
C6H4-4-OCH3
fur-2-yl
thien-2-yl
H
1m
2c
2d
2e
2j
H
CH3
CH3
CH3
CH3
CH3
66 ( 7
ND
ND
2k
ND
Ro 15-1788
clonazepam
0.90 ( 0.05
0.85 ( 0.02
0.90
1.97
15 ( 5
100 ( 10
a
Percentages of inhibition of specific [3H]Ro 15-1788 binding at 10 µM compound concentration are means ( SEM of five determinations.
Ki values are means ( SEM of three determinations. c GABA ratio ) (Ki without GABA)/(Ki with GABA). The effects of the compounds
b
d
at 0.5 µM on TBPS binding were normalized with respect to the corresponding action of clonazepam. The data represent the means (
standard error of three separate experiments. e Not determined.
Ta ble 4. Biological Activity of Selected Triazinobenzimidazole
Derivatives
or hydroxyl), exhibited Ki values of 44 and 74 nM,
respectively. No less surprising was the fact that two
substituents at the 3′ and 4′ positions of the phenyl ring
can be associated with submicromolar affinity or lack
of affinity, depending on whether they are two hydroxyls
(1i) or methoxyls (1h ), respectively.
anticonvulsant action: proconvulsant
diazepam
no.
ED50 (mg/kg)a
actionb
antagonismc
1c
1e
1k
2c
no effectd
no effectd
no effectd
60e
no effectd
no effectd
no effectd
no effectd
no effectd
no effectd
no effectd
no effectd
The lack of affinity shown by compounds 1d ,g,h was
unexpected on the basis of the alignment reported in
Figure 1, where the 4′ position of triazinobenzimidazole
1c matches the 2 position of pyridodiindole III. If this
latter position bears a chlorine, a methyl, or a methoxyl,
the affinity of the resulting pyridodiindole derivative
does not change significantly.29,30 Extending our com-
parisons to 2-arylpyrazoloquinolines I, it is well-known
that hydrogen, chlorine, and methoxyl at the 4′ position
of the pendant phenyl ring (spatially close to the 4′
position of 1c in Figure 1) are reported to confer
subnanomolar affinity,6 while the effects on potency
produced by a 4′-methyl unfortunately have not been
disclosed in the literature, although this structure was
included in two patents.31,32
The GABA ratio values for the most active compounds
showed a trend of the efficacy profile of partial inverse
agonists/antagonists for series 1 (values ranging be-
tween 0.87 and 1.25) and of a partial agonist for the
10-methyl derivative 2c, with a value of 1.70.
Compounds 1c,e,f,j,k and 2c were also assayed with
the [35S]TBPS shift test,33,34 which confirmed the ef-
ficacy profile predicted by the GABA ratio values, since
compounds 1c,e,f,j,k showed [35S]TBPS shift values
ranging between 5 and 35, similar to the value of 15
obtained for the antagonist Ro 15-1788, and compound
2c showed a value of 66, intermediate between those of
the full agonist clonazepam (fixed here at a value of 100)
and Ro 15-1788. The agonist activity of the N(10)-methyl
diazepam
Ro 15-1788
â-CCM
0.4e
no effectf
no effectf
no effectf
19.4e
0.3e
0.02e
a
Dose necessary to antagonize the convulsant action of PTZ
b
(80 mg/kg, sc) in 50% of mice. Dose necessary to induce convul-
sions in 50% of the mice that had previously been given
a
subconvulsant dose of PTZ (40 mg/kg, sc). c Dose necessary to
antagonize the anticonvulsant effect of diazepam (2.5 mg/kg, ip)
in mice that had been given a convulsant dose of PTZ (80 mg/kg,
d
sc). The highest concentration of the compounds tested was 250
mg/kg. e Values represent the means of at least three determina-
tions (e20% differences between experiments). f The highest dose
administered of Ro 15-1788 and â-CCM (3-carbomethoxy-â-car-
boline51) was 100 and 30 mg/kg, respectively.
derivative 2c, unable to form the hydrogen bond at the
A2 subsite (Figure 1), is not in contradiction with Cook’s
model,17 which considers the interaction at the A2
subsite a necessary requirement only for inverse agonist
activity.
P h a r m a cologica l Stu d ies. The pharmacological
profile of compounds 1c,e,k and 2c was evaluated by
in vivo assays carried out as described previously.35 The
results obtained (Table 4) showed no in vivo efficacy for
compounds 1c,e,k , which were neither anticonvulsant
nor proconvulsant, nor did they antagonize the anti-PTZ
action of diazepam. Their lack of in vivo efficacy might
be due either to a poor penetration through the blood-
brain barrier or to an unfavorable pharmacokinetics.
Compound 2c showed an anticonvulsant activity with
an ED50 of 60 mg/kg and a complete lack of diazepam

100 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Primofiore et al.
antagonism or proconvulsant action. For this compound,
the in vivo activity was consistent with the in vitro
results, as the GABA ratio of 1.70 and the TBPS shift
of 66 were predictive of an in vitro partial agonist
efficacy.
the 10-methylated product 2c and the 1-methylated 7,
while compounds 1d ,g,h gave almost exclusively the
1-methylated products 8-10, respectively. Though we
are aware that the product of a chemical reaction
depends not only on thermodynamic but also on kinetic
factors, the results of these methylations seemed to
suggest that for 1c the forms mainly involved in the
tautomeric equilibria are Va (active) and Vc (inactive),
while for compounds 1d ,g,h the equilibrium seemed
mostly to involve the inactive form Vc.
Although the conditions of the binding studies and
those of the NMR and methylation experiments are
quite different, the methylation reaction results are in
agreement with our binding hypothesis postulating the
N(10)-H function (tautomer Va ) as an essential phar-
macophore element and with the binding affinity data.
NMR- a n d Meth yla tion Rea ction -Ba sed Stu d ies
of Ta u tom er ic Equ ilibr ia . The puzzling SARs ob-
served in the series of triazinobenzimidazoles 1 could
not be rationalized in terms of substituent effects or by
comparison with structures of classic BzR ligands (see
molecular alignment in Figure 1). Therefore, NOE
difference experiments were performed on 1c,d ,g,h in
DMSO-d6, to investigate tautomeric equilibria involving
these compounds which could be potentially related to
their binding affinities. Upon irradiation of the hydrogen
of 1c at δ 14.28, NOE enhancements were observed for
H(9) at δ 7.67 and H(2′) at δ 8.07 of the fused benzene
and pendant phenyl rings, respectively. The NOE effect
observed for H(9) is consistent with its proximity to the
N(10)-H of the imidazole moiety (structure Va ), while
the NOE enhancement observed for H(2′) can be at-
tributed to the enolic tautomer Vb featuring a hydroxy
group at the 4 position. A similar pattern of NOE effects
was obtained from the 4′-methyl derivative 1g.
Con clu sion s
Sound SARs could not be deduced by simple qualita-
tive analysis of the results obtained for this new class
of BzR ligands. Insertion of an electron-donating group
(OCH3, OH, CH3) at the 4′ position of the 3-phenyl ring
of 1c gave either highly potent products (1e,f) or a
compound devoid of any affinity (1g). Also the insertion
at the same 4′ position of a group with opposite
electronic effects such as a chlorine (1d ) completely
abolished affinity.
When the NMR- and methylation-based studies of
tautomeric equilibria are taken into consideration,
contrasting results are again observed. While NMR
studies revealed the same tautomeric equilibria (Va ,b)
for 1c (active) and 1g (inactive) and a different tautomer
(Vc) for inactive 1d ,h , methylation reactions suggested
the equilibrium form Va only for 1c and the tautomer
form Vc for 1d ,g,h .
Although at present we do not know the reasons for
these conflicting results, the higher affinity shown by
some 3-aryltriazinobenzimidazoles V with respect to the
N-benzylindolylglyoxylylamides IV (R ) H) confirmed
the rationale of our design, which was that constraining
the indolylglyoxylyl fragment would enhance the bind-
ing affinity.
When the same experiments were performed on the
4′-chloro and 3′,4′-dimethoxy derivatives 1d ,h , none of
the above NOE effects were detected, suggesting that
these compounds exist only in the tautomeric form Vc
featuring the N(1)-H on the triazinone nucleus.
The tautomeric forms of 1c,d ,h could explain the
binding affinities observed, since the N(10)-H function
is an essential pharmacophore element (see Figure 1).
However, the tautomeric behavior exhibited by 1g
(devoid of affinity), which appeared to be identical to
that of 1c (highly potent), seems not to be in agreement
with this assumption.
The lack of correlation between the binding affinities
observed and the tautomeric equilibria which emerged
from the NMR studies in DMSO solution prompted us
to investigate this problem from another point of view,
in an indirect, even if less rigorous, way. Taking into
account that methylation products might have furnished
useful information about the tautomer(s) prevalently
present in solution, methylation reactions were carried
out on compounds 1c,d ,g,h with methyl iodide in hot
ethanol, in the presence of sodium ethylate (Scheme 2).
Compound 1c gave, in an approximately 1:2 ratio, both
Exp er im en ta l Section
Ch em ist r y. Melting points were determined using
a
Reichert Ko¨fler hot-stage apparatus and are uncorrected.
Infrared spectra were obtained on a PYE/UNICAM model PU
9561 spectrophotometer in Nujol mulls. Nuclear magnetic
resonance spectra were recorded in DMSO-d6 solutions with
a Bruker AC 200 or a Varian CFT-20 spectrometer using TMS
as an internal standard. Mass spectra were obtained on a
Hewlett-Packard 5988 A spectrometer using a direct injection
probe and an electron beam energy of 70 eV. Magnesium
sulfate was always used as the drying agent. Evaporations
were made in vacuo (rotating evaporator). Analytical TLC was
carried out on Merck 0.2-mm precoated silica gel aluminum
sheets (60 F-254). Silica gel 60 (230-400 mesh) was used for
column chromatography. Elemental analyses were performed
by our Analytical Laboratory and agreed with theoretical
values to within (0.4%.
3-Su bstitu ted [1,2,4]Tr iazin o[4,3-a ]ben zim idazol-4(10H)-
on es 1a -c,e-h ,j,m a n d 2c,e,g,h ,j (P r oced u r e 1). A solution
of 2-hydrazinobenzimidazole (3)19 or 2-hydrazino-1-methyl-
benzimidazole (4)19 (4 mmol) and the appropriate glyoxylic
acid36-40 (4.4 mmol) in 10 mL of ethanol was refluxed for 2 h.
After cooling, the precipitate which formed was collected to
give the substituted (benzimidazol-2-ylhydrazono)acetic acids

3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 101
5a -c,e-h ,j,m and 6c,e,g,h ,j, which were purified by suspen-
sion in hot methanol (Table 1, Supporting Information). They
can be cyclized by means of the following methods.
Meth od A. The acid derivatives 5c,e and 6c (2 mmol) were
heated at a temperature of 30-40 °C above their melting
points for 10 min.
Meth od B. A suspension of the acid derivatives 5f-h ,j,m
and 6e,g,h ,j (2 mmol) in 5 mL of absolute ethanol, at 0 °C,
was saturated with anhydrous HCl and then refluxed for 4-7
h, monitoring the reaction by TLC analysis. The reaction
mixture was evaporated to dryness and the solid residue was
treated with saturated sodium hydrogen carbonate aqueous
solution.
Meth od C. A suspension of the acid derivatives 5a ,b (2
mmol) in 20 mL of glacial acetic acid was refluxed for 1 h.
The solution obtained was evaporated to dryness and the oily
residue was filtered on a silica gel chromatographic column
(eluting system: toluene:chloroform ) 1:1).
81%; mp 188-190 °C. Anal. Calcd for C18H16N4O3 (Supporting
Information).
From the mother liquor it was not possible to isolate the
10-methyl isomer 2d , 2g, or 2h , respectively, of which only
traces were present (TLC analysis).
Bin d in g Stu d ies. [3H]Ro 15-1788 (specific activity 83.2 Ci/
mmol) and [35S]TBPS (specific activity 80 Ci/mmol) were
obtained from DuPont de Nemours, New England Nuclear
Division (Dreieichenhaim, Germany). All other chemicals were
of reagent grade and obtained from commercial suppliers.
Bovine cerebral cortex membranes were prepared in ac-
cordance with ref 43. The membrane preparations were
subjected to a freeze-thaw cycle, washed by suspension,
centrifuged in 50 mM tris-citrate buffer pH 7.4 (T1), and then
used in the binding assay. Protein concentration was assayed
by the method of Lowry et al.44
[3H]Ro 15-1788 and [35S]TBPS binding studies were per-
formed as previously reported.35
In Vivo Stu d ies. Proconvulsant, anticonvulsant, and di-
azepam antagonism actions were performed as previously
reported.35
All the crude solid products obtained were purified by
recrystallization from the appropriate solvent (Table 2, Sup-
porting Information).
NMR-Ba sed Stu d ies of Ta u tom er ic Equ ilibr ia . Samples
were prepared by dissolving compounds 1c,d ,g,h in DMSO-
d6. 1H NMR spectra and NOE difference experiments were
recorded on a Bruker AMX-500 spectrometer, at 300 K. Signals
were referenced to the residue solvent signal at 2.5 ppm.
Molecu la r Mod elin g. Molecular modeling was performed
using the software package SYBYL45 running on a Silicon
Graphics R8000 Indigo 2 workstation. Models of the 4′-
chlorophenylpyrazoloquinoline derivative of type I and the
2-chloropyridodiindole derivative of type III correspond to
crystal structures retrieved from the October 1995 release
(5.10 version for UNIX platforms) of the Cambridge Structural
Database46 (codes COVLOO and J OJ HIZ, respectively). The
model of triazinobenzimidazole 1c was constructed using the
SYBYL fragment library and geometry-optimizing the trial
conformation by the semiempirical quantum-mechanics AM1
method47 available within the MOPAC program.48 MOPAC
was run under default settings, using the keyword “MMOK”.
Superimpositions of the molecular models were performed as
described in previous works.18,35
3-Su bstitu ted [1,2,4]Tr iazin o[4,3-a ]ben zim idazol-4(10H)-
on es 1d ,k ,l a n d 2d ,k (P r oced u r e 2). A solution of 3 or 4 (4
mmol) and the appropriate glyoxylic acid ethyl ester39,41,42 (4.4
mmol) in 10 mL of absolute ethanol was refluxed for 2-11 h
(TLC analysis). After cooling, the precipitate which formed was
collected and purified by recrystallization from the appropriate
solvent (Table 2, Supporting Information).
3-(3′,4′-Dih yd r oxyp h en yl)-[1,2,4]t r ia zin o[4,3-a ]b en z-
im id a zol-4(10H)-on e (1i). A solution of boron tribromide (6
mmol) in 1 mL of anhydrous dichloromethane was added
dropwise, at -10 °C, under stirring and in a nitrogen atmo-
sphere, to a suspension of the dimethoxy derivative 1h (0.9
mmol) in 8 mL of the same solvent. Stirring was continued
for 30 min at -10 °C and for 1 h at room temperature. The
reaction mixture was carefully added dropwise to 5 mL of
methanol at -10 °C. The suspension obtained was evaporated
to dryness and the solid residue was treated with saturated
sodium hydrogencarbonate aqueous solution. The crude prod-
uct 1i was collected and purified by recrystallization (Table
2, Supporting Information).
Ack n ow led gm en t. This work was supported by
grants from the Ministry of University and Scientific
and Technological Research (MURST) (research fund
60%) and from the Italian National Research Council
(Progetto di Ricerca CNR: Chimica Fine II).
Rea ction of 3-P h en yl-[1,2,4]tr ia zin o[4,3-a ]ben zim id a -
zol-4(10H)-on e (1c) w ith Meth yl Iod id e: 3-P h en yl-1-
m eth yl-[1,2,4]tr ia zin o[4,3-a ]ben zim id a zol-4(1H)-on e (7)
a n d 3-P h en yl-10-m eth yl-[1,2,4]tr ia zin o[4,3-a ]ben zim id a -
zol-4(10H)-on e (2c). A solution of methyl iodide (0.4 mL, 7
mmol), sodium ethoxide (0.16 g, 7 mmol of sodium in 15 mL
of absolute ethanol), and 1c (0.35 g, 1.4 mmol) was refluxed
for 12 h. After cooling, the precipitate which formed was
collected and purified by recrystallization from DMF to give
0.20 g (yield 52%) of pure 7, mp 196-197 °C. Anal. Calcd for
Su p p or tin g In for m a tion Ava ila ble: Tables containing
the IR, 1H NMR, and MS spectral data of compounds
5a -c,e-h ,j,m and 6c,e,g,h ,j (Table 1), compounds 1a -l and
2c-e,g,h ,j,k (Table 2), and compounds 7-10 (Table 3). This
material is available free of charge via the Internet at http://
pubs.acs.org.
C
16H12N4O (Supporting Information).
The reaction mother liquor was evaporated to dryness and
the residue obtained was suspended in water and neutralized
with dilute hydrochloric acid to give crude 2c, which was
recrystallized from DMF (0.110 g, yield 29%).
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102 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
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J M991131H