1,2,3-Triazolo [1,5-a][1,4]- and 1,2,3-triazolo[l,5-a]-[1,5]benzodiazepine derivatives: Synthesis and benzodiazepine receptor binding

Article abstract of DOI:10.1016/S0014-827X(98)00025-1

This paper reports the synthesis of new 1,2,3-triazolo[1,4]benzodiazepine and 1,2,3-triazolo[1,5]benzodiazepine derivatives and their evaluation toward benzodiazepine receptors. Receptor affinity gradually and remarkably increases by moving the nitrogen atom of the central ring from position 3 through 4 to position 5, to give the most effective compound 6a (K(i) = 150 nM). N-methylation of the diazepine ring (7a) lowers receptorial binding. Introduction of a chlorine atom on the benzene ring doubles the K(i) value (6b) which remains unaltered by the N-methylation (7b).

Full text of DOI:10.1016/S0014-827X(98)00025-1

Il Farmaco 53 (1998) 305–311
1,2,3-Triazolo[1,5-a][1,4]- and 1,2,3-triazolo[1,5-a]-
[1,5]benzodiazepine derivatives: synthesis and benzodiazepine
receptor binding
a
Lucia Bertelli a, Giuliana Biagi a, Irene Giorgi a, Oreste Livi a, , Clementina Manera ,
*
Valerio Scartoni a, Claudia Martini b, Gino Giannaccini b, Letizia Trincavelli b, Pier Luigi Barili c
a Dipartimento di Scienze Farmaceutiche, Facolta di Farmacia, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy
`
`
b Istituto Policattedra di Discipline Biologiche, Facolta di Farmacia, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy
`
`
c Dipartimento di Chimica Biorganica, Facolta di Farmacia, Universita di Pisa, Via Bonanno 33, 56126 Pisa, Italy
`
`
Received 6 October 1997; accepted 4 March 1998
Abstract
This paper reports the synthesis of new 1,2,3-triazolo[1,4]benzodiazepine and 1,2,3-triazolo[1,5]benzodiazepine derivatives and their
evaluation toward benzodiazepine receptors. Receptor affinity gradually and remarkably increases by moving the nitrogen atom of the central
ring from position 3 through 4 to position 5, to give the most effective compound 6a (Kis150 nM). N-methylation of the diazepine ring
(7a) lowers receptorial binding. Introduction of a chlorine atom on the benzene ring doubles the Ki value (6b) which remains unaltered by
the N-methylation (7b).
q 1998 Elsevier Science S.A. All rights reserved.
Keywords: 1,2,3-Triazolo[1,5-a][1,4]benzodiazepine; 1,2,3-Triazolo[1,5-a][1,5]benzodiazepine; Benzodiazepine receptor binding
1. Introduction
The benzodiazepine receptor (BZR) is a cell-surface
GABA receptor/chloride ion channel complex, which can
bind either classical benzodiazepines or several other com-
pounds with very different structures [1–7]. The interaction
of these compounds can produce a continuum of intrinsic
activity ranging from full agonists (sedative–hypnotic, anx-
iolytic, anticonvulsant and myorelaxant agents) to antago-
nists (devoid of pharmacological efficacy) or to inverse
agonists (with proconvulsant, convulsant and anxiogenic
properties). Considerable efforts have been directed toward
the characterization of the essential features for ligand–BZR
interaction, assuming that agonists, antagonists and inverse
agonists bind to the same binding domain of the BZR. How-
ever, some pharmacophoric descriptors (hydrogen donors,
hydrogen acceptors, and lipophilic groups) must differ to
allow different efficacy [8].
which had shown a moderate affinity toward benzodiazepine
receptors, theoretical calculations based upon the SAR sug-
gested introduction of a substituent which generates a region
of negative molecular electronic potential (MEP) on theben-
zene ring.
As a route to directly introduce substituents on the benzo-
fused ring, we considered the nitration reaction of the tri-
azolobenzodiazepine moiety; unfortunately, the nitration
failed since the starting tricyclic system was recovered
unreacted or it underwent oxidative demolition under
stronger reaction conditions. Moreover, attempts of a prelim-
inary substitution on the 2-aminophenylacetic acid, the pre-
cursor of the azide, which represents the key intermediate for
the synthesis of the tricyclic system, failed because of facile
intramolecular cyclization (under acid conditions) to 2-
oxyindoline.
In a previous paper [9] concerning a series of 1,2,3-tri-
azolo[1,5-a][1,3]benzodiazepine derivatives (formula A)
* Corresponding author.
0014-827X/98/$19.00 q 1998 Elsevier Science S.A. All rights reserved.
PII S0014-827X(98)00025-1

306
L. Bertelli et al. / Il Farmaco 53 (1998) 305–311
Thus, we realized the synthesis of isomeric new 1,2,3-
(chlorine) or with an acetyl group, respectively, to give the
triazolo[1,5-a][1,4]benzodiazepines (formula B) and
1,2,3-triazolo[1,5-a][1,5]benzodiazepines (formula C).
corresponding substituted tricyclic analogues.
2. Chemistry
The 2-cyanophenylazide [10] reacted with diethyl or
dimethyl acetylenedicarboxylate in refluxing benzene to give
the expected triazole diesters 1a and 1b (Scheme 1). Reduc-
tion of the cyano group to amine directly provided the
corresponding tricyclic derivatives 3-substituted-1,2,3-tri-
azolo[1,5-a][1,4]benzodiazepin-4-ones 2a and 2b in good
yield. Alkylation of 2a with methyl iodide gave the corre-
sponding N-methyl derivative 3a.
The synthetic route which leads to the new 1,2,3-tri-
azolo[1,5]benzodiazepines bearing a substituent on the ben-
zene ring is described in Scheme 2. Thus, starting from 2-
nitrophenylazide [12] and diethyl acetonedicarboxylate,
according to the literature [11], the 1-(2-nitrophenyl)-4-
carbethoxy-5-carbethoxymethyl-1H-1,2,3-triazole (4a), the
corresponding 2-amino-phenyltriazole 5a and the known 3-
carbethoxy-1,2,3-triazolo[1,5-a][1,5]benzodiazepin-5-one
(6a) were synthesized.
In the first case, the 2-cyanophenylazide [10] allowed the
preparation of one triazolo[1,4]benzodiazepine derivative,
unsubstituted on the benzene ring (Scheme 1). In fact, some
attempts to introduce substituents on the benzene ring, start-
ing from 2-cyanoaniline substituted with nitro or bromo
groups to give the appropriate substituted 2-cyanophenyla-
zides and/or the corresponding tricyclic derivatives, failed.
On the other hand, the3-carbethoxy-1,2,3-triazolo[4,5-a]-
[1,5]benzodiazepin-5-one had been described in the litera-
ture [11] starting from 2-nitrophenylazide, so that the same
synthetic route (Scheme 2) could be employed from 2-nitro-
phenylazides previously substituted with a halogen atom
Scheme 1. Synthesis of 1,2,3-triazolo[1,5-a][1,4]benzodiazepine derivatives.
Scheme 2. Synthesis of 1,2,3-triazolo[1,5-a][1,5]benzodiazepine derivatives.

L. Bertelli et al. / Il Farmaco 53 (1998) 305–311
307
1
Similarly, by reacting 2-nitro-4-chlorophenylazide [13]
or 2-nitro-4-acetylphenylazide [14] with diethyl acetonedi-
carboxylate, the expected triazole derivatives 4b and 4c were
obtained in 25% and 18% yields, respectively. These target
compounds were obtained in low yields because the main
reaction product was the corresponding4-substitutednitroan-
iline, probably resulting from the cleavage of the formed
1,2,3-triazole in an alkaline medium. Even using mild
experimental conditions, the azide decomposed into benzo-
furazane N-oxide, and polymeric or tarry material was also
recovered.
Reduction of the nitro group of the triazole derivative 4a
was achieved by catalytic hydrogenation according to Smal-
ley and Teguiche [11], while reduction of the analogues 4b
and 4c was satisfactorily carried out with iron powder, in
order to preserve the chloro and acetyl substituents. The ami-
nophenyl 1,2,3-triazole esters 5a–c obtained by intramolec-
ular cyclization (in boiling xylene in the presence of a
catalytic amount of p-toluenesulfonic acid (5a) [11] or boil-
ing toluene in the presence of catalytic concentrated sulfuric
acid for 5b and 5c) were converted into the corresponding
tricyclic derivatives 6a–c. Treatment of the latter compounds
with methyl iodide provided the corresponding N-methyl
derivatives 7a–c in good yields.
recorded on a Perkin-Elmer model 1310 spectrometer. H
NMR spectra (internal standard tetramethylsilane (TMS))
were recorded on a Varian CFT 20 or a Bruker AC 200
¨
spectrometer operating at 80 MHz or at 200 MHz, respec-
tively. 13C NMR spectra were recorded with the Bruker AC
¨
200 spectrometer at 50 MHz. Chemical shifts assignments
were confirmed, when possible, with HETCOR experiments.
Mass spectra were performed with a Hewlett Packard MS/
System 5988. Thin-layer chromatography (TLC) data were
obtained with Riedel de Haen, 37360 DC-Karten F254, 0.2
mm, eluting with an AcOEt/petroleum ether 1:1 mixture.
Elemental analyses (C, H, N) were within "0.4% of the
theoretical values and were performed on a Carlo Erba ele-
mental analyzer model 1106 apparatus. Column chromatog-
raphy was performed on silica gel 60 (230–400 mesh).
Petroleum ether corresponds to the fraction boiling at 40–
608C.
3.1.1. General procedure for the synthesis of 1-(2-cyano-
phenyl)-1H-1,2,3-triazole-4,5-dicarboxylates 1a–b
A solution of 2-cyanophenylazide (3.00 g, 20.83 mmol)
and 34.0 mmol of diethyl or dimethyl acetylenedicarboxylate
in 90 ml of anhydrous benzene was heated under reflux for
20 h. The solvent wasevaporatedinvacuoandtheoilyresidue
underwent a short distillation at 1508C/5 mmHg to distil off
the excess diethyl acetylenedicarboxylate. The distillation
residue consisted of solidified 1a or 1b.
Compound 1a (5.90 g, yield 90%) could be purified by
distillation at 2258C/0.2 mmHg; m.p. 59–618C; IR (nujol):
n (cmy1) 2240 (C^N), 1730 and 1230 (COOR). Anal.
(C15H14N4O4): C, H, N.
The structures of all the newly prepared compounds were
assigned according to the reaction mechanismsand werecon-
firmed by analytical and spectroscopic data. 13C NMR data
of the tricyclic compounds are reported in Table 1.
3. Experimental
Compound 1b (4.91 g, yield 83%) could be purified by
crystallization from MeOH; m.p. 104–1068C; IR (nujol): n
(cmy1) 2240 (C^N), 1730 and 1240 (COOR); MS (m/z):
286 (Mq), 227, 168, 155, 143, 102. Anal. (C13H10N4O4):
C, H, N.
3.1. Chemistry
Melting points were determined on a Kofler hot-stage
apparatus and are uncorrected. IR spectra in nujol mulls were
Table 1
13C NMR data (d, ppm) for compounds 2, 3, 6 and 7 in DMSO
2a
3a
6a
6b
6c
7a
7b
7c
C-4
C-4a
C-5
C-6
C-7a
C-7
140.1
134.1
157.4
41.2
132.4
128.7
129.9
129.5
122.9
135.1
159.6
61.0
13.7
–
139.8
134.9
155.8
49.1
134.5
129.0
130.6
129.8
122.6
134.8
159.5
61.0
13.6
33.9
–
135.9
134.4
30.7
167.6
130.1
122.7
130.1
125.1
123.6
126.3
160.2
60.6
13.9
–
135.8
134.5
30.7
167.5
131.3
122.0
134.1
125.3
124.9
125.3
160.1
60.7
13.8
–
137.8
134.7
30.7
137.1
134.4
31.0
166.3
134.4
123.9
130.5
126.5
124.2
127.5
160.3
60.8
14.0
36.5
–
136.1
134.6
31.0
166.8
134.8
124.2
137.1
126.5
125.5
126.7
160.3
60.8
14.0
36.5
–
138.0
134.5
30.8
167.5
130.2
122.4
136.2
124.6
124.1
129.2
160.1
60.7
13.7
–
196.3
26.5
166.5
134.8
123.9
137.2
125.7
124.3
130.5
160.3
60.7
13.8
36.5
196.5
26.7
C-8
C-9
C-10
C-10a
COO
OCH2
CH3
NCH3
CO-8
CH3
–
–
–
–
–
–
–
–
–

308
L. Bertelli et al. / Il Farmaco 53 (1998) 305–311
3.1.2. 5,6-Dihydro-4-oxo-4H-1,2,3-triazolo[1,5-a][1,4]-
benzodiazepine-3-carboxylates 2a–b
Isolation of 4a: benzofurazane N-oxide [12]: 0.300 g,
yield 15%, m.p. 69–718C; 2-nitroaniline: 0.930 g, yield 45%,
m.p. 71–728C; 4a [11]: 1.00 g, yield 19%; m.p. 78–798C.
Isolation of 4b: 6-chlorobenzofurazane N-oxide [13]:
0.700 g, yield 27%, m.p. 46–478C; 2-nitro-4-chloroaniline:
0.920 g, yield 35%, m.p. 128–1298C; 4b: 1.430 g; yield 25%;
m.p. 121–1228C (EtOH); IR (nujol): n (cmy1) 1730 and
1220 (COOR); MS (m/z): 382 (Mq), 337, 280, 253, 207,
180, 127; 1H NMR (CDCl3, 80 MHz): d (ppm) 8.11 (d, 1H,
H-39), 7.74 (dd, 1H, H-59), 7.49 (d, 1H, H-69), 4.38 and
4.04 (2q, 4H, 2OCH2), 3.91 (s, 2H, CH2CO), 1.37 and 1.15
(2t, 6H, 2CH3). Anal. (C15H15N4O6Cl): C, H, N.
Aqueous Ni-Raney suspension ((1 g) and ammonium
hydroxide (32%, 2 ml) were added to a solution of 1a or 1b
(7.0 mmol) in methanol (150 ml), and the mixture was
hydrogenated at room temperature and pressure. The suspen-
sion was heated under reflux for 1 h, the catalyst was filtered
off, washed with hot methanol and the combined filtrates
evaporated in vacuo to give the title compounds.
2a: 1.81 g, yield 95%; m.p. 242–2448C (EtOH); IR
(nujol): n (cmy1) 3230 (NH), 1660(C_O), 1725and1220
(COOR); MS (m/z): 272 (Mq), 243, 229, 197, 171, 125,
116; 1H NMR (DMSO, 200 MHz): d (ppm) 9.09 (bt, 1H,
NH), 8.00 (m, 1H, H-10), 7.68–7.58 (m, 3H, H-7, H-8 and
H-9), 4.37 (q, 2H, Js7.1 Hz, OCH2), 4.27 (d, 2H, CH2-6),
1.32 (t, 3H, CH3). 13C NMR: see Table 1. Anal.
(C13H12N4O3): C, H, N.
Isolation of 4c: 6-acetylbenzofurazane N-oxide [14]:
0.147 g, yield 5.5%, m.p. 89–908C; 2-nitro-4-acetylaniline:
1.447 g, yield 54%, m.p. 143–1458C; 4c: 1.070 g, yield 18%;
m.p. 134–1358C (EtOH); IR (nujol): n (cmy1) 1730 and
1230 (COOR), 1690 (C_O); MS (m/z): 390 (Mq), 261,
1
2b: 1.35 g, yield 75%; m.p. 270–2728C (MeOH); IR
(nujol): n (cmy1) 3200 (NH), 1720 and 1220 (COOR),
1650 (C_O); MS (m/z): 258 (Mq), 229, 197, 129. Anal.
(C12H10N4O3): C, H, N.
215, 187, 161, 127; H NMR (CDCl3, 80 MHz): d (ppm)
8.68 (d, 1H, H-39), 8.32 (dd, 1H, H-59), 7.72 (d, 1H, H-69),
4.45 and 4.11 (2q, 4H, 2OCH2), 4.00 (s, 2H, CH2CO), 2.73
(s, 3H, CH3CO), 1.44 and 1.21 (2t, 6H, 2CH3). Anal.
(C17H18N4O7): C, H, N.
3.1.3. Ethyl 5,6-dihydro-5-methyl-4-oxo-4H-1,2,3-tri-
azolo[1,5-a][1,4]benzodiazepine-3-carboxylate 3a
3.1.5. Ethyl 1-(2-aminophenyl)-4-carbethoxy-1H-1,2,3-
triazole-5-acetate 5a
The compound was prepared as described in the literature
[11] from 4a (0.440 g, 1.26 mmol) by catalytic hydrogen-
ation: 0.390 g, yield 97%; m.p. 100–1028C (EtOH).
Methyl iodide (1.2 ml, 19.3 mmol) was added toa solution
of 2a (0.300 g, 1.10 mmol) in 70 ml of ethanolic 0.1%
sodium hydroxide (1.75 mmol). The mixture was stirred at
room temperature for 22 h. The mixture was neutralized with
10% hydrochloric acid, concentrated in vacuo, treated with
H2O and extracted with CHCl3. The organic layer, dried
(MgSO4) and evaporated, provided 3a: 0.280 g, yield 89%;
m.p. 166–1688C (EtOH); IR (nujol): n (cmy1) 1720 and
1210 (COOR), 1650 (C_O); MS (m/z): 286 (Mq), 257,
185, 156, 128, 116; 1H NMR (DMSO, 200 MHz): d (ppm)
8.02 (m, 1H, H-10), 7.72 (m, 1H, H-8), 7.67 (m, 1H, H-7),
7.62 (m, 1H, H-9), 4.53 (s, 2H, CH2-6), 4.37 (q, 2H, Js7.1
Hz, OCH2), 3.11 (s, 3H, NCH3), 1.33 (t, 3H, CH3). 13C
NMR: see Table 1. Anal. (C14H14N4O3): C, H, N.
3.1.6. General procedure for the synthesis of ethyl 1-(2-
aminoaryl)-4-carbethoxy-1H-1,2,3-triazole-5-acetates5b–c
5% FeCl3 solution (0.3 ml) and iron powder (0.350 g)
were added to a solution of 4b or 4c (1.70 mmol) in aqueous
ethanol (60%, 60 ml). The mixture was heated under reflux
for 3 h. The hot reaction mixture was filtered and the filtrate
concentrated in vacuo. The residue was treated with H2O and
the suspension obtained was alkalinized with 32% ammo-
nium hydroxide solution and extracted with CHCl3. The
organic layer was dried (MgSO4) and evaporated to give the
title compounds.
5b: 0.556 g, yield 93%; m.p. 105–1078C (EtOH); IR
(nujol): n (cmy1) 3460 and 3340 (NH2), 1720 and 1250
(COOR); MS (m/z): 352 (Mq), 265, 250, 222, 205, 177,
165, 142; 1H NMR (CDCl3, 80 MHz): d (ppm) 7.03 (d, 1H,
H-69), 6.84 (d, 1H, H-39), 6.79 (dd, 1H, H-59), 4.43 and
4.11 (2q, 4H, 2OCH2), 3.91 (s, 2H, CH2CO), 1.42 and 1.21
(2t, 6H, 2CH3). Anal. (C15H17N4O4Cl): C, H, N.
5c: 0.589 g, yield 95%; m.p. 105–1078C (EtOH). IR
(nujol): n (cmy1) 3400 and 3330 (NH2), 1730 and 1220
(COOR), 1690 (C_O); MS (m/z): 360 (Mq), 315, 273,
247, 230, 213, 185, 171, 143; 1H NMR (CDCl3, 80 MHz):
d (ppm) 7.43–7.20 (m, 4H, H-29, H-39, H-59 and H-69), 4.44
and 4.20 (2q, 4H, 2OCH2), 3.94 (s, 2H, CH2CO), 2.59 (s,
3H, CH3CO), 1.43 and 1.21 (2t, 6H, 2CH3). Anal.
(C17H20N4O5): C, H, N.
3.1.4. General procedure for the synthesis of ethyl 4-
carbethoxy-1-(2-nitroaryl)-1H-1,2,3-triazole-5-acetates
4a–c
40 ml of ethanolic sodium ethoxide solution (0.350 g, 15.2
g atoms sodium) were slowly added dropwise ((1 h) to a
stirred and cooled (0–58C) suspension of the suitable 2-
nitrophenylazide (15.0 mmol) and diethyl acetonedicarbox-
ylate (3.0 ml, 16.5 mmol) in 40 ml of absolute ethanol. The
suspension was stirred at room temperature for one night
(4a), for 5 h (4b) or for one night at 0–58C (4c), then it
was concentrated in vacuo (temperature F458C), treated
with H2O and extracted with AcOEt. The organic layer, after
washing with NaCl solution, was dried (MgSO4) and evap-
orated to give a solid residue which underwent flash-chro-
matography through a silica gel column ((14=4 cm).
Elution with petroleum ether/AcOEt 3:1 and successively
2:1 provided the following results.

L. Bertelli et al. / Il Farmaco 53 (1998) 305–311
309
3.1.7. Ethyl 5,6-dihydro-5-oxo-4H-1,2,3-triazolo-
[1,5-a][1,5]benzodiazepine-3-carboxylate 6a
(t, 3H, CH3). 13C NMR see Table 1. Anal. (C14H14N4O3):
C, H, N.
The compound was prepared as described in the literature
[11] from 5a (0.390 g, 1.22 mmol) by refluxing in 25 ml of
xylene in the presence of a catalytic amount of p-toluenesul-
fonic acid: 0.265 g, yield 80%; m.p. 194–1958C (EtOH); 1H
NMR (DMSO, 200 MHz): d (ppm) 10.49 (bs, 1H, NH),
7.97 (dd, 1H, J9,10s8.02 Hz, J8,10s1.50 Hz, H-10), 7.59
(ddd, 1H, J8,9s7.56 Hz, H-8), 7.42 (ddd, 1H, J7,9s1.44
Hz, H-9), 7.37 (dd, 1H, J7,8s7.82 Hz, H-7), 4.39 (q, 2H,
Js7.1 Hz, OCH2), 4.14 (s, 2H, CH2-5), 1.36 (t, 3H, CH3).
13C NMR see Table 1. Anal. (C13H12N4O3): C, H, N.
7b: 0.235 g, yield 92%; m.p. 150–1528C (EtOH); IR
(nujol): n (cmy1) 1720 and 1210 (COOR), 1670 (C_O);
1
MS (m/z): 320 (Mq), 235, 218, 191, 189, 155, 124; H
NMR (DMSO, 200 MHz): d (ppm) 7.93 (d, 1H, J9,10s8.68
Hz, H-10), 7.82 (d, 1H, J7,9s2.23 Hz, H-7), 7.58 (dd, 1H,
H-9), 4.39 (q, 2H, Js7.1 Hz, OCH2), 3.30 (s, 2H, CH2-5),
3.19 (s, 3H, NCH3), 1.36 (t, 3H, CH3). 13C NMR see Table
1. Anal. (C14H13N4O3Cl): C, H, N.
7c: 0.210 g, yield 80%; m.p. 152–1548C (DMF–H2O); IR
(nujol): n (cmy1) 1710 and 1250 (COOR), 1680 (C_O);
1
MS (m/z): 328 (Mq), 257, 243, 199, 187, 158, 130; H
NMR (DMSO, 200 MHz): d (ppm) 8.15 (m, 1H, H-10),
8.06 (m, 2H, H-7 and H-9), 4.41 (q, 2H, Js7.1 Hz, OCH2),
3.36 (s, 2H, CH2-5), 3.20 (s, 3H, NCH3), 1.36 (t, 3H, CH3).
13C NMR see Table 1. Anal. (C16H16N4O4): C, H, N.
3.1.8. General procedure for the synthesis of 8-substituted-
5,6-dihydro-5-oxo-4H-1,2,3-triazolo[1,5-a][1,5]benzo-
diazepine-3-carboxylates 6b–c
One drop of 98% sulfuric acid was added to a solution of
5b or 5c (1.50 mmol) in toluene (50 ml) and heated under
reflux for 4 h. After cooling, the solution was washed with
H2O, dried (MgSO4) and evaporated in vacuo to give the
title compounds.
4. Biological
6b: 0.446 g, yield 97%; m.p. 268–2708C (AcOEt); IR
(nujol): n (cmy1) 3180 (NH), 1710 and 1250 (COOR),
1670 (C_O); MS (m/z): 306 (Mq), 250, 204, 178, 165,
124; 1H NMR (DMSO, 200 MHz): d (ppm) 10.58 (bs, 1H,
NH), 8.00 (d, 1H, J9,10s8.65 Hz, H-10), 7.48 (dd, 1H,
J7,9s2.32 Hz, H-9), 7.42 (d, 1H, H-7), 4.39 (q, 2H, Js7.1
Hz, OCH2), 4.17 (s, 2H, CH2-5), 1.36 (t, 3H, CH3). 13C
NMR see Table 1. Anal. (C13H11N4O3Cl): C, H, N.
6c: 0.461 g, yield 98%; m.p. 214–2168C (EtOH); IR
(nujol): n (cmy1) 3160 (NH), 1710 and 1240 (COOR),
1660 (C_O); MS (m/z): 314 (Mq), 269, 258, 243, 185,
169, 141, 130; 1H NMR (DMSO, 200 MHz): d (ppm) 10.62
(bs, 1H, NH), 8.10 (d, 1H, J9,10s8.24 Hz, H-10), 7.94 (dd,
1H, J7,9s1.85 Hz, H-9), 7.92 (d, 1H, H-7), 4.40 (q, 2H,
Js7.1 Hz, OCH2), 4.18 (s, 2H, CH2-5), 2.63 (s, 3H,
COCH3), 1.36 (t, 3H, CH3). 13C NMR see Table 1. Anal.
(C15H14N4O4): C, H, N.
The prepared tricyclic compounds 2a–b, 3a, 6a–c and 7a–
c were tested for their ability to inhibit benzodiazepinerecep-
tor binding, by measuring the concentration able to displace
the [3H] Ro 15-1788 from bovine brain membranes.
The experimental details of the receptor binding assays
were reported in a previous paper [15].
Furthermore, the GABA ratio values of active compounds
were evaluated as an in vitro indicator of the agonist, inverse-
agonist or antagonist properties [16].
5. Results and discussion
3.1.9. Ethyl 5,6-dihydro-6-methyl-5-oxo-4H-1,2,3-triazolo-
[1,5-a][1,5]benzodiazepine-3-carboxylate 7a, ethyl
8-chloro-5,6-dihydro-6-methyl-5-oxo-4H-1,2,3-triazolo-
[1,5-a][1,5]benzodiazepine-3-carboxylate 7b and ethyl
8-acetyl-5,6-dihydro-6-methyl-5-oxo-4H-1,2,3-triazolo-
[1,5-a][1,5]benzodiazepine-3-carboxylate 7c
Methyl iodide (0.6 ml, 9.60 mmol) was added to a stirred
solution of the triazolobenzodiazepine 6a, 6b or 6c (0.80
mmol) in 0.5% NaOH ethanolic solution (10 ml), and stir-
ring was continued at room temperature for 3 h. Afterdilution
with H2O the title compounds precipitated as a white solid
which was collected by filtration and washed with H2O.
7a: 0.190 g, yield 83%; m.p. 183–1858C (EtOH);1HNMR
(DMSO, 200 MHz): d (ppm) 7.93 (m, 1H, H-10), 7.72 (m,
2H, H-7 and H-8), 7.53 (m, 1H, H-9), 4.39 (q, 2H, Js7.1
Hz, OCH2), 3.29 (s, 2H, CH2-5), 3.27 (s, 3H, NCH3), 1.35
The results in Table 2 show that affinity toward the
benzodiazepine receptors of the 1,2,3-triazolo[1,5-a]-
benzodiazepine derivatives gradually and remarkably
increases ((10-fold), by moving the nitrogen atom of the
central ring from position 3 through 4 to position 5; in fact,
the 3-carbethoxy-1,2,3-triazolo[1,5-a][1,3]benzodiazepin-
5-one, the most effective derivative previously prepared [9],
showed an inhibition constant Kis1600 nM, while the
[1,4]benzodiazepine isomer 2a shows Kis908 nM and the
other [1,5]benzodiazepine isomer 6a shows Kis150 nM. In
all cases the GABA ratio values ((1.2) indicate a antagonist
action.
Moreover, comparison of these three isomers, which are
members of three heterocyclic series, shows that the N-meth-
ylation of the diazepine ring lowers the receptor binding (see

310
L. Bertelli et al. / Il Farmaco 53 (1998) 305–311
Table 2
Inhibition of [3H] Ro 15-1788 binding and GABA ratio
Comp.
Ki (nM) a
GABA ratio b
2a
2b
3a
6a
6b
6c
7a
7b
7c
908"81
1.19
0.60
–
1.26
1.55
–
1.12
1.93
–
1190"97
2600"240
150"11
348"35
7700"755
931"91
354"31
)8000
The tests were carried out using dimethyl sulfoxide (2%) as solvent unless
otherwise stated.
a Ki represents the mean"SEM of three determinations.
b GABA ratiosKi(compound)/Ki(compoundq50 mM GABA) per-
formed in three experiments.
Fig. 1. Interactions of 6a with A2 (H-bond acceptor site), H1 and H2 (H-
bond donor sites) and L1 (lipophilic region).
the N-methyl derivative of the previous [1,3] series [9] and
compounds 3a and 7a).
It is worth noting for the [1,4]benzodiazepine series that
the methyl ester 2b is less effective than the ethyl ester 2a
and the GABA ratio value (0.60) suggests an inverse-agonist
efficacy.
Regarding the derivatives of the [1,5]benzodiazepine
series (compounds 6a–c and 7a–c), which interact better
with the receptor site than the corresponding 1,3- and 1,4-
derivatives, introduction of an 8-substituent on the benzo-
fused ring decreases the receptor affinity.
However, introduction of a chlorine atom maintains good
activity (compounds 6b and 7b), whilst the acetyl group
strongly decreases the activity (compounds 6c and 7c). The
chloro-substituted compounds 6b (Kis348 nM) and thecor-
responding N-methyl derivative 7b (Kis354 nM) show
equivalent affinities and the GABA ratio values (1.55 and
1.93, respectively) suggest a partial-agonist/agonistactivity.
These considerations indicate that the presence of a sub-
stituent such as Cl or COCH3 influences receptor binding
probably by electronic and steric effects.
Moreover, the recently proposed inclusive pharmacophore
model of agonist/inverse-agonist binding sites [8] allows us
to evaluate the interactions of the functional groups present
on these structures. The possible and indicative interactions
of the more active ligands 6a, 6b and 7b are illustrated in
Figs. 1 and 2. Fig. 1 shows the most active 8-unsubstituted
compound 6a: the NH function may be able to form an H-
bond with the A2 site; the CO amide function may be able to
accept an H-bond from the H1 site; likewise, a nitrogen atom
of the triazole ring may accept an H-bond from the H2 site;
the ethyl group may be arranged in the lipophilic region L1.
All these interactions can agree with an antagonist/inverse-
agonist activity. Similarly, the receptor binding hypothesized
for 8-chloro-substituted 6b and 7b, which possess similar
affinity, is reported in Fig. 2. This result indicates that in this
case N-methylation did not influence the affinity, therefore
the NH function is not involved in the receptor binding in the
Fig. 2. Interactions of 6b or 7b with L1 and L2 (lipophilic regions) and H1
and H2 (H-bond donor sites); NH or NCH3 functions are not involved in
the A2 but in the L3 region.
A2 area. Thus, according to this hypothesis, 6b and 7b can
occupy the lipophilic areas L1 and L2 with the 8-chlorobenzo-
fused moiety and interact with the H1 and H2 donor sites
through the triazole nitrogen atoms and the carbonyl func-
tions, respectively. This arrangement can agree with the ago-
nist activity of 6b and 7b; in fact, the A2 interaction site,
which is characteristic of inverse-agonist/antagonist effi-
cacy, is not involved. The NH or NCH3 groups fall in the L3
lipophilic area, which together with the L2 one, is essential
for agonist biological function. Occupation of the L3 region
justifies the higher GABA ratio of the NCH3 derivative 7b
with respect to the N-unsubstituted 6b.

L. Bertelli et al. / Il Farmaco 53 (1998) 305–311
311
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Acknowledgements
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