Benzodiazepine receptor ligands. 4. Synthesis and pharmacological evaluation of 3-heteroaryl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5- oxides

Article abstract of DOI:10.1021/jm981126y

The synthesis of new 3-heteroaryl-8-chloropyrazolo[5,1- c][1,2,4]benzotriazine 5-oxides and their binding activities at the central benzodiazepine receptor (BZR) are reported. The derivatives substituted at the 3-position with electron-rich five-membered

Full text of DOI:10.1021/jm981126y

2218
J . Med. Chem. 1999, 42, 2218-2226
Ben zod ia zep in e Recep tor Liga n d s. 4. Syn th esis a n d P h a r m a cologica l
Eva lu a tion of 3-Heter oa r yl-8-ch lor op yr a zolo[5,1-c][1,2,4]ben zotr ia zin e 5-Oxid es
Annarella Costanzo,*,§ Gabriella Guerrini,§ Giovanna Ciciani,§ Fabrizio Bruni,§ Silvia Selleri,§ Barbara Costa,†
Claudia Martini,† Antonio Lucacchini,† Petra Malmberg Aiello,‡ and Alessandra Ipponi‡
Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Firenze, Via Gino Capponi 9, 50121 Firenze, Italy,
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` degli Studi di Pisa, Via Bonanno 6,
56126 Pisa, Italy, and Dipartimento di Farmacologia Preclinica e Clinica Aiazzi-Mancini, Universita` degli Studi di Firenze,
Viale Pieraccini 6, 50139 Firenze, Italy
Received December 10, 1998
The synthesis of new 3-heteroaryl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides and their
binding activities at the central benzodiazepine receptor (BZR) are reported. The derivatives
substituted at the 3-position with electron-rich five-membered rings, such as pyrrole 11,
2-thiophene 13c, or 3-thiophene 13d , showed good affinity values for BZR. In in vivo tests the
3-(thien-3-yl)-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (13d ) showed selective anti-
convulsant activity.
In tr od u ction
hydrogen bond. The 5-oxide group also has an important
role in reinforcing this hydrogen bond.
As part of our program directed toward the search
for central nervous system (CNS) agents, the synthesis
and evaluation of the affinity on the central benzo-
diazepine receptor (BZR) of numerous 2-, 3-, 7-, and
8-substituted pyrazolo[5,1-c][1,2,4]benzotriazines and
corresponding 4- and 5-oxides have been reported.1-4
From binding data we pointed out that some pyrazolo-
[5,1-c][1,2,4]benzotriazine 5-oxides, bearing at the 3-po-
sition an ethoxycarbonyl group or bromine and at the
8-position a chlorine or small lipophilic group such as
ethoxy or methyl, were new ligands for BZR with good
affinity values (Ki range 35-93 nM) and with an efficacy
trend typical of agonists and partial agonists (GR ratio
2.67-1.31).2 In particular the 3-(ethoxycarbonyl)-8-
chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (I) was
the derivative with the highest affinity (35.0 ( 2.0 nM)
and agonist profile (GR 1.91). In general, modification
of the ethoxycarbonyl group at the 3-position, such as a
nitrile, carboxy, or carboxyamide group, or its replace-
ment by a six-membered ring or a nitro group was found
to be detrimental to receptor binding, suggesting the
importance of an ester group for the receptor fitting. At
present all results seem to indicate that the 3-substitu-
ent is critical for the BZR affinity.2-4 On the basis of
structure-activity relationship (SAR) data, we have
proposed a pharmacophore/receptor model for these new
BZR ligands. In this model the pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide binds to BZR with N-1 and N-4
by means of a hydrogen bond involving the H2 and H1
donor receptor sites, respectively. The substituent at the
3-position fits into the lipophilic region L1/L2. In par-
ticular, if this substituent is an ethoxycarbonyl group,
as in the lead compound I, the lone pair orientation of
the carbonyl oxygen of the estereal chain can reinforce
the receptor binding by means of a three-centered
The lead compound I, selected for in vivo testing,
showed poor anticonvulsant and anxiolytic activity and
no myorelaxant properties.2 These in vivo pharmaco-
logical results, in contrast with binding data, might be
attributed to bioavailability problems or easy metabolic
cleavage of the carboxylic ester to biologically inactive
acid.
To obtain improved efficacy and greater selectivity in
vivo, we decided on isosteric replacement of the ester
function by 1,2,4-oxadiazole, 1,2,4-triazole, and isoxazole
rings in the lead compound.5-7 These small heterocycles,
in which a nitrogen or oxygen takes the place of the
carbonyl oxygen, might mimic an ester group and if
tolerated in the 3-position could give metabolically more
stable derivatives.
Moreover, we decided to replace the ester group with
other heterocycles such as 2- or 3-thiophene and pyrrole
which are different from the five-membered rings cited
above for electronic and steric features. The synthesis
of the new series of 3-heteroaryl-8-chloropyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxides seems very useful either
for SAR or to further confirm our hypothesis about the
critical role of the substituent at the 3-position of these
ligands for binding at BZR.
Ch em istr y
All pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides de-
scribed here are listed in Table 1. To obtain 8-chloro-
pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides bearing in
the 3-position, as isosteric replacement of the ester
function, the triazolo and oxadiazole moiety, several
synthetic pathways were followed5 (see Scheme 1).
The starting material for synthesis of both derivatives
was the 3-carbamoyl-8-chloropyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide (1)1 that by treatment with dimethyl-
formamide-dimethyl acetal (DMF-DMA) or dimethyl-
acetamide-dimethyl acetal (DMA-DMA)5 yielded the
intermediate acylamidine 2a or 2b, respectively. These
intermediates were cyclized with hydrazine hydrate at
* Corresponding author. Tel: +39-55-2757296. Fax: +39-55-240776.
E-mail: Costanzo@farmfi.scifarm.unifi.it.
§ Universita` degli Studi di Firenze, Via Gino Capponi 9.
† Universita` degli Studi di Pisa, Via Bonanno 6.
‡ Universita` degli Studi di Firenze, Viale Pieraccini 6.
10.1021/jm981126y CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/02/1999

Benzodiazepine Receptor Ligands
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2219
Ta ble 1. Chemical Data for Pyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxides
compd
R3
formula (MW)
mp (°C) (recrystallization solvent) yield
2a
2b
3a
3b
4a
4b
5b
6
7
9
10
11
13c
-CONdCH(NMe2)
-CONdC(NMe2)Me
-1,2,4-triazol-3-yl
-1,2,4-triazol-5-Me-3-yl
-1,2,4-triazol-1(2)(4)-Me-3-yl
-1,2,4-triazol-1(2)(4),5-diMe-3-yl
-1,2,4-oxadiaxol-3-Me-5-yl
-CONHCHO
C13H11N6O2Cl (318.75)
C14H13N6O2Cl (332.78)
259-261 (washed by ether) 97%
234-235 (washed by ether) 73%
>300 (ethanol) 53%
C
C
C
C
C
C
C
C
11H6N7OCl (287.69)
12H8N7OCl (301.72)
12H8N7OCl (301.72)
13H10N7OCl (315.75)
12H7N6O2Cl (302.70)
11H6N5O3Cl (291.67)
11H7N6O3Cl (306.69)
14H14N5O3Cl (335.78)
>300 (methoxyethanol) 20%
273-276 (acetic acid) 20%
279-280 (acetic acid, 50%) 25%
249-250 (methoxyethanol) 80%
271-273 (washed by water) 77%
262-263 (methoxyethanol) 60%
199-200 (ethanol/water) 97%
244-246 (ethanol) 57%
210-211 (methoxyethanol) 90%
225-226 (methoxyethanol) 58%
203-204 (methoxyethanol) 35%
220-221d (methoxyethanol) 80%
244-245 (methoxyethanol) 75%
224-225 (water) 90%
-CONdCHNHOH
-NHCOOtBu
-NH2
C9H6N5OCl (235.65)
-pyrrol-1-yl
C
C
13H8N5OCl (295.71)
13H7N4OSCl (302.75)
-2-thienyl
a
13c′
-2-thienyl
C13H7N4SCl (286.75)
13H7N4OSCl (302.75)
13d
16
17
18
19
20
-3-thienyl
C
-CH2CONH2
C11H8N5O2Cl (277.69)
C11H7N4O3Cl (278.67)
C13H12N5O2Cl (305.75)
-CH2COOH
-C(CHO)dCHNMe2
-C(CHO)dCHOH
-4-isoxazolyl
220-222 (washed with water) 60%
212-213 (water) 50%
266-267 (methoxyethanol) 35%
C
C
12H7N4O2Cl (274.68)
12H6N5O2Cl (287.68)
a
This compound is a 5-deoxy derivative: 3-(thien-2-yl)-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine.
Sch em e 1a
a
(a) DMF-DMA or DMA-DMA; (b) hydrazine hydrate; (c) CH3I; (d) acetic acid/H2O; (e) NH2OH‚HCl.
90-100 °C to obtain the 3-(1,2,4-triazol-3-yl) derivatives
3a and 3b, which were then alkylated by methyl iodide/
dimethylformamide on N-1(2)(4) atom to yield the
methyl derivatives 4a and 4b.
anhydride or in POCl3. If compound 2a is treated with
H2O/AcOH, the 3-N-formylamide derivative 6 is ob-
tained, by the obvious hydrolysis of the acylamidine
group. Both these products (6 and 7) were included in
the SAR for the BZR because they bear at the 3-position
substituents with particular electronic and polarity
features.
The same intermediate acylamidines, 2a and 2b, were
also used to obtain the 3-(1,2,4-oxadiazol-5-yl) deriva-
tives by treatment with hydroxylamine hydrochloride.
In this case only compound 2b yielded the desired
oxadiazole derivative 5b, while compound 2a gave only
the corresponding 3-N-hydroxylaminomethyleneamide
7 (see Scheme 1), whose cyclization failed either in acetic
Scheme 2 depicts the route used to prepare the
3-(pyrrol-1-yl) derivative 11. Treatment of 3-carboxy-8-
chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide1 (8) with
diphenyl phosphorazidate (DPPA)/triethylamine/tert-

2220 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costanzo et al.
Sch em e 2a,b
a
b
(a) Et3N/DPPA; (b) acetic acid/hydrochloric acid; (c) dimethoxytetrahydrofuran. (a) EtOH/hydrochloric acid as catalyst; (b) 10% aq
NaOH.
Sch em e 3a
a
(a) EtOH; (b) sulfuric acid; (c) 10% aq NaOH; (d) H2SO4/NaNO2; (e) POCl3/Me2NCHO; (f) 20% aq NaOH; (g) NH2OH‚HCl.
butyl alcohol8 gave the 3-tert-butoxycarbamoylamino
derivative 9. The attempt to obtain 9 by another route
with lead tetraacetate/tert-butyl alcohol9 failed. Com-
pound 9 was easily hydrolyzed with hot 12 M HCl and
AcOH to the 3-amino derivative 10. The 3-amino-8-
chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (10)
was converted to the 3-(pyrrol-1-yl) derivative 11 by
treatment with 2,5-dimethoxytetrahydrofuran in acetic
acid.
Scheme 3). As a precursor of the isoxazole ring, the
malondialdehyde moiety was created at the 3-position
of the pyrazolo[5,1-c][1,2,4]benzotriazine system. In fact
this function is known to be very versatile and can
cyclize to produce a large variety of aromatic as well as
nonaromatic systems.12-14
The starting material to achieve the key intermediate,
3-(1-oxo-2-hydroxyvinyl)-8-chloropyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (19), was the 3-(carboxymethyl)-
8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (17)
whose synthesis was rather laborious. The 2-nitro-5-
chlorophenylhydrazine was reacted with 2-oxosuccino-
nitrile (see Experimental Section) to obtain the 1-(2-
nitro-5-chlorophenyl)-5-aminopyrazole-4-acetonitrile (14).
The acidic hydrolysis of this latter compound yielded
the corresponding 4-acetamide derivative 15, which was
cyclized to benzotriazine system 16 in alkali medium.
Treatment with concentrated H2SO4/NaNO2 gave the
desired 3-carboxymethyl derivative 17 which was,
through the use of Vilsmeier-Haack reagent, trans-
formed into 3-enamine 18 and after hydrolysis into
3-malondialdehyde derivative 19. This latter compound
The 2-nitro-5-chlorophenylhydrazine1 was reacted
with 2-(thien-2-yl)- and 2-(thien-3-yl)-3-oxapropane-
nitrile10,11 (c,d ) to obtain the aminopyrazoles bearing,
at the 4-position, a 2-thienyl or 3-thienyl ring: 12c and
12d , respectively. These aminopyrazoles (12c and 12d ),
in turn, were cyclized to the benzotriazine system in
alkaline medium,1-4 achieving the desired compounds
pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 13c and 13d
(see Scheme 2). Compound 13c, 3-(thien-2-yl)-8-chloro-
pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide, was deoxi-
dated to compound 13c′, 3-(thien-2-yl)-8-chloropyrazolo-
[5,1-c][1,2,4]benzotriazine, by treatment with triethyl
phosphite(TEP)/toluene3 to give us further information
for the SAR.
1
exhibits an interesting H NMR spectra run either in
A laborious synthetic path was used to obtain com-
pound 20 bearing at the 3-position an isoxazole ring (see
CDCl3 or in DMSO-d6. In CDCl3, two signals as a broad
singlet at 7.20 ppm for OH proton and at 9.20 ppm for

Benzodiazepine Receptor Ligands
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2221
Ta ble 2. BZR Ligand Affinity of Pyrazolo[5,1-c][1,2,4]benzotriazine 5-Oxides
compd
2a
R3
% inibitiona
Ki (nM)b
GRc
-CONdCHNMe2
-CONdC(NMe2)Me
-1,2,4-triazol-3-yl
-1,2,4-triazol-5-Me-3-yl
-1,2,4-triazol-1(2)(4)-Me-3-yl
-1,2,4-triazol-1(2)(4),5-diMe-3-yl
-1,2,4-oxadiazol-3-Me-5-yl
-CONHCHO
48.9 ( 1.32
58.5 ( 2.53
44.4 ( 3.59
15.2 ( 0.98
0
2b
3a
3b
4a
4b
5b
6
0
63.5 ( 4.57
85.5 ( 6.89
0
844.2
2.07
7
9
-CONdCHNHOH
-NHCOOtBu
0
10
11
13c
13c′
13d
20
Ie
-NH2
33.9 ( 1.50
87.5 ( 6.30
82 ( 6.20
66 ( 4.50
100 ( 6.70
66.0 ( 2.1
99.8 ( 5.0
-pyrrol-1-yl
61.5 ( 3.50
10.3 ( 0.81
1.24
1.54
-2-thienyl
d
-2-thienyl
-3-thienyl
36.3 ( 2.11
1.23
-4-isoxazolyl
-COOEt
35.0 ( 0.2e
10 ( 1.1
1.91e
1.5
diazepam
a
b
Percent inhibition values of specific [3H]Ro15-1788 binding at 10 µM concentration are means ( SEM of five determinations. Ki
values are means ( SEM of five determinations. c GR (GABA ratio) ) IC50 compound + 10 µM GABA, performed in five independent
experiments. 5-Deoxy derivative. e See ref 2.
d
olefinic proton were seen; the aldehydic proton was
evidenced only after D2O treatment at 9.4 ppm, while
the singlet at 7.20 ppm exchanges with the same
treatment. In DMSO-d6 the aldehydic and olefinic
protons were seen as a broad singlet (8.7 ppm).
Finally, treatment of 19 with hydroxylamine hydro-
chloride in ethanol gave the desired compound 3-(isox-
azol-4-yl)-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-ox-
ide (20). The same compound 19 could be the starting
material to obtain the 3-(furan-3-yl) derivative, if treated
with ethyl bromopropionate in alkali medium,12 but in
our case the synthesis failed.
These new ligands (11, 13c, and 13d ) bind to BZR
with N-1 and N-4 by means of a hydrogen bond
involving H2 and H1 donor receptor site as lead com-
pound I.4 In particular for these 3-heteroaryl deriva-
tives, a π-π stacking interaction between the receptor
and the substituent at the 3-position, thiophene, and
pyrrole, which are π-excessive rings, can be hypoth-
esized. These rings, acting as a π-electron donor, might
take place in a limited size lipophilic pocket in the
lipophilic region L1/L2.15-17 From literature it is well-
recognized that the sulfur atom of a thiophene ring is
substantially weak to form a hydrogen bond with an
active proton.16 Consequently the sulfur atom of com-
pounds 13c and 13d cannot form a three-centered
hydrogen bond as can the carbonyl group of the lead
compound I. The enhanced affinity of ligand 13c with
respect to 13d might be explained by a better accom-
modation of the thienyl ring of the 2-isomer than the
3-isomer into the lipophilic pocket reinforcing the π-π
stacking interaction. The important role of the 5-oxide
group reinforcing the hydrogen bond (N-4/H1 receptor)
is confirmed by 5-deoxy derivative 13c′, which is com-
pletely inactive (compare 13c and 13c′).
Resu lts a n d Discu ssion
Biologica l Resu lts. The BZR binding affinity of
pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides was evalu-
ated by their ability to displace [3H]Ro15-1788 from its
specific binding in bovine brain membranes. Binding
data for all new compounds for lead compound I are
reported in Table 2. As can be observed from these
results, the reaction intermediates 2a , 2b, 6, 7, 9, and
10, which have at the 3-position hydrophilic or bulkier
groups than the ethoxycarbonyl moiety in lead com-
pound I, display no or very weak affinity to BZR. The
3-triazolyl, 3-oxadiazolyl, and 3-isoxazolyl derivatives
3a , 3b, 4a , 4b, 5b, and 20 lack BZR affinity, excluding
their bioisosterism with the 3-ethoxycarbonyl group.
Interestingly, in derivatives 11, 13c, and 13d the
replacement of the ester group of lead compound I by
electron-rich five-membered rings, such as pyrrole or
2- or 3-thiophene, retains significant affinity to BZR.
In fact the Ki values (61.5 nM for 11, 36.3 nM for 13d ,
and 10.3 nM for 13c) indicate a lower, comparable to,
and higher binding affinity respectively than that lead
compound I (Ki 35.0 nM). Significantly the 3-(thien-2-
yl)-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine (13c′), deoxy
analogue derivative of 13c, is devoid of affinity.
Compounds 3a , 3b, 4a , 4b, 5b, and 20, bearing at the
3-position a rigid isoster moiety of ester function,
probably do not correctly accommodate the 3-substituent
into the lipophilic pocket and consequently cannot
contribute to form the three-centered hydrogen bonding
with nitrogen or oxygen. In addition, oxadiazole, tri-
azole, and isoxazole rings, which are more electron-poor
than pyrrolo and thiophene, are unable to favor a π-π
stacking interaction.
P h a r m a cologica l Resu lts. Compounds 13c and 13d
were chosen for in vivo testing. Muscle relaxant, anti-
convulsant, and anxiolytic activities of these substances
were evaluated in comparison to those of vehicle, lead
compound I, and diazepam (used as positive control) (see

2222 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costanzo et al.
Ta ble 3. Muscle Relaxant, Anticonvulsant, and Anxiolytic Effects of 13c, 13d , and I in Comparison with Diazepam
muscle relaxant effect
rota-rod test
anticonvulsant activity
antianxiety activity
light/dark box
n of crosses
66 16.4 ( 0.9
against PTZ-
induced attacks
against MES-induced
hindlimb extensionb
n of falls from
treatmenta
CMC, 1%
mg/kg
n
rotating rod
%
n
%
% lethality
27.8
n
% time in light
0.1 mL 113
0.24 ( 0.05
0.23 ( 0.09
0.56 ( 0.18
0.73 ( 0.28*
1.4 ( 0.4***
0.4 ( 0.16
11.5
36
5.5
100 ( 4.2
diazepam
0.1
0.3
1
3
100
21
16
15
10
15
14
47.6***
81.2***
100***
100***
0
8
22.0 ( 2.7*
95.5 ( 11.0
10
90***
10
0*
13 28.5 ( 3.87*** 144.9 ( 9.2***
10 100***
10 16.5 ( 3.6
10 15.2 ( 1.7
211.1 ( 29.9***
114.8 ( 8.8
flumazenil
flumazenil + diazepam 100
0.3
0.46 ( 0.2
14.2∧∧
flumazenil + diazepam 100
10 18.6 ( 1.3∧
104.1 ( 11.7∧
1
13c
3
10
30
100
300
100
0.3
100
1
10
30
10 15.0 ( 2.1
12 20.0 ( 1.7
13 17.6 ( 2.5
13 18.4 ( 2.2
88.9 ( 6.8
122.2 ( 6.4*
115.5 ( 11.2
114.5 ( 7.8
10
12
8
0 ( 0
10
11
10
12
0
0
0
18.2
20
8.3*
0.08 ( 0.08
0.2 ( 0.2
0.5 ( 0.31
16.6
12.5
50
13c + diazepam
13c + diazepam
13d
10
12
0.17 ( 0.1∧
100
13 26.6 ( 3.2
130.9 ( 7.3
11
13
13
11
0.43 ( 0.3
0.08 ( 0.08
0.15 ( 0.1
0.64 ( 0.24
18.2
14
18
11
14.3
33.3*
27.3
28.6
22.2
18.2
12 16.3 ( 2.0
12 19.0 ( 2.9
13 15.3 ( 2.0
94.2 ( 11.8
104.3 ( 11.3
107.4 ( 7.0
61.5***
46.1***
36.4∧
100
30
13d + flumazenil
100
30
100
300
I
20
32
20
15
28.1*
25
9
12
0
0
22.2
0**
10 19.1 ( 2.7
11 15.2 ( 1.8
106.2 ( 10.7
124.6 ( 10.8*
0.5 ( 0.15
0.33 ( 0.18
a
b
Treatment with compounds 13c, 13d , 21, and diazepam was performed 30 min and flumazenil 40 min before the test. Maximal
electroshock (MES) ) 40 mA, 0.2 s, 50 Hz. Tonic hindlimb extension was considered as end point. *P < 0.05, **P < 0.01, ***P < 0.001
versus control mice; ∧P < 0.05, ∧∧P < 0.001 versus 13d - and diazepam-treated mice, respectively.
Table 3). During the 30-min postdrug period, prior to
test, the animals were carefully observed for gross
behavior in their home cages. No behavior alterations
were noticed following the administration of each of the
substances under study.
strong attacks with a high degree of mortality observed
in the MES test.
As for anxiolytic effects, diazepam was again used as
reference molecule under our conditions. It demon-
strated a dose-related effect at doses of 1 and 3 mg/kg
po. Among the tested compounds, only the lead molecule
I showed any anxiolytic effect at the same dose (100 mg/
kg po) at which it demonstrated anticonvulsant effect.
Compound 13c provided neither muscle relaxant nor
anticonvulsant activity, and its anxiolytic activity was
very weak, but it was able (at a dose of 100 mg/kg po)
to antagonize diazepam (1 mg/kg po)-induced falls from
rotating rod, thus demonstrating its partial agonist
profile.
None of the newly synthesized molecules, including
the lead compound, which were tested in a wide range
of doses, caused a statistically significant impairment
in rota rod performance, thus indicating a total absence
of any muscle relaxant effect, while diazepam, from 1
mg/kg po up, significantly diminished the mice’s motor
coordination.
Anticonvulsant activity was studied using two differ-
ent kinds of convulsant stimuli: pentylenetetrazole
(PTZ) for chemically induced convulsion and maximal
electroshock (MES) for electrically induced ones. Diaze-
pam was able to prevent both kinds of convulsion,
although to a lesser extent those of MES. The lead
molecule showed slight anticonvulsant activity only in
the PTZ-induced attacks at the dose of 100 mg/kg po
and significantly decreased, like diazepam, the MES-
induced lethality.
Su m m a r y
In conclusion, the introduction in the 3-position of a
pyrazolobenzotriazine system of an electron-rich ring,
as 3-thienyl, yielded compound 13d which is endowed
with a good affinity (Ki 36.3 nM) and in vivo selective
pharmacological profile. This derivative seems to be the
most interesting because it manifested a selective
anticonvulsant activity without affecting the animals’
motor coordination. This effect is probably due to its
agonist/antagonist characteristic according to its GABA
ratio value (1.23), so that a specific GABAA/BZ receptor
subtype might be selectively activated.18,19
Unexpectedly the 2-thienyl isomer 13c, having the
best affinity (Ki 10.3 nM) in vitro, shows very weak
intrinsic efficacy in vivo. The lack of activity in vivo of
13c versus 13d could be explained by different meta-
bolic oxidation pathways of the thiophene ring in the
On the other hand 13d , even if it has a lower GABA
ratio (1.23) with respect to I (1.91) and 13c (1.54),
prevented PTZ-induced convulsion in a statistically
highly significant manner at doses of 30 and 100 mg/
kg po. Flumazenil, an antagonist on the BZR, at a dose
of 100 mg/kg ip was able to significantly antagonize the
protective effects of both 13d (30 mg/kg) and diazepam
(0.3 mg/kg). Similarly to diazepam, 13d yielded anti-
convulsant activity also in the MES test. The minor
efficacy of both the molecules is probably due to very

Benzodiazepine Receptor Ligands
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2223
two isomers,20 also considering that the conjugation of
the heteroaryl ring with pyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide system is different in the 3-thienyl than
the 2-thienyl ring.
The results of this work have further focused on the
critical role of the 3-substituent of the pyrazolo[5,1-c]-
[1,2,4]benzotriazine 5-oxide ligands: it could fit in a
lipophilic pocket into the lipophilic region of the receptor
protein, which probably has a different size in the
GABAA/BZ subtype, affecting both ligand potency and
intrinsic activity (efficacy), according to reported litera-
ture data.18,19
mL) was added hydrazine hydrate (0.30 mmol, 0.05 mL), and
the reaction was kept at 90-100 °C for 2 h. The residue was
filtered and recrystallized from a suitable solvent.
3-(1,2,4-Triazol-3-yl)-8-chloropyrazolo[5,1-c][1,2,4]benzotri-
a zin e 5-Oxid e, 3a . From 2a ; orange crystals. IR ν cm-1
3000-2500, 1600, 1510. H NMR (DMSO-d6) δ: 8.69 (s, 1H,
H-2); 8.56 (bs, 1H, CH triazole); 8.46 (d, 1H, H-6); 8.42 (d, 1H,
H-9); 7.80 (dd, 1H, H-7). Anal. (C11H6N7OCl) C, H, N.
:
1
3-((1,2,4-Tr ia zol-5-m eth yl)-3-yl)-8-ch lor op yr a zolo[5,1-
c][1,2,4]ben zotr ia zin e 5-Oxid e, 3b. From 2b; orange crys-
tals. IR ν cm-1: 3280, 1600, 1540. 1H NMR (DMSO-d6) δ: 8.63
(s, 1H, H-2); 8.44 (d, 1H, H-6); 8.38 (d, 1H, H-9); 7.78 (dd, 1H,
H-7); 2.42 (s, 3H, CH3). Anal. (C12H8N7OCl) C, H, N.
Gen er a l P r oced u r e for Syn th esis of 4a a n d 4b. A
suspension of compound 3a or 3b (0.35 mmol) in anhydrous
DMF (15 mL) was added to a suspension of 50% NaH (0.76
mmol, 18.2 mg) and an excess of CH3I (0.05 mL) and was kept
at 50-60 °C for 18 h. The final solution was evaporated and
the residue purified by recrystallization with 50% AcOH.
Exp er im en ta l Section
The structures of all compounds were supported by their
IR spectra (KBr pellets in Nujol mulls, Perkin-Elmer 681
spectrophotometer) and 1H NMR data (measured with a
Varian Gemini at 200 MHz, chemical shifts expressed in δ
(ppm) using DMSO-d6 or CDCl3 as solvent). Melting points
were determined with a Gallenkamp apparatus and were
uncorrected. Elemental analyses were performed by the
laboratories of Dipartimento Farmaco-Chimico-Tecnologico of
Universita` di Siena, Italy, with a Perkin-Elmer, model 240C,
elemental analyzer, and their results are within (0.4% of
theoretical values. The purity of samples was determined by
means of TLC, which was performed using Machery-Nagel
Duren, Alugram silica gel plates. The MS spectra were
obtained with a QMD-1000 apparatus (Carlo Erba, Milano,
Italy) after GC-MS analysis. GC experimental conditions:
column SBP-5, i.d. 0.25 mm, l 30 m, gradient temperature
elution at 50 °C, 2 min, 15 °C/min up to 280 °C. MS analysis:
EI, 70 eV.
2-Oxosu ccin on itr ile. A suspension of 50% NaH in mineral
oil (200 mmol, 4.8 g) was added to anhydrous toluene (300 mL)
in a 1-L round-bottomed flask. After addition of tert-amyl
alcohol (2 mL) the mixture was heated at 70 °C, and a solution
of succinonitrile (100 mmol, 8 g) and ethyl formate (100 mmol,
8.5 mL) in anhydrous toluene (50 mL) was added dropwise in
1 h. After 6 h of stirring and heating at 70-80 °C, the resulting
solid was allowed to stand overnight. The mixture was treated
with ice-water (400 mL), and the aqueous phase was sepa-
rated and washed with diethyl ether. Acidification with 12 M
HCl causes the separation of an oil which was extracted with
diethyl ether. The extracts were dried on anhydrous Na2SO4
and evaporated to dryness, and a red oil was obtained. The
final product was identified by GC-MS spectra m/z (%): 79
(25.68), 53 (82.88), 40 (100).
3-((1,2,4-Tr ia zol-1(2)(4)-m eth yl)-3-yl)-8-ch lor op yr a zolo-
[5,1-c][1,2,4]ben zotr ia zin e 5-Oxid e, 4a . From 3a ; orange
1
crystals. IR ν cm-1: 1600, 1520. H NMR (CDCl3) δ: 8.62 (s,
1H, H-2); 8.48 (m, 2H, H-6, H-9); 8.14 (s, 1H, CH triazole);
7.58 (dd, 1H, H-7); 4.03 (s, 3H, NCH3). Anal. (C12H8N7OCl) C,
H, N.
3-((1,2,4-Tr ia zol-1(2)(4),5-d im eth yl)-3-yl)-8-ch lor op yr a -
zolo[5,1-c][1,2,4]b en zot r ia zin e 5-Oxid e, 4b . From 3b ;
orange crystals. IR ν cm-1: 1600, 1520. 1H NMR (CDCl3) δ:
8.58 (s, 1H, H-2); 8.50 (d, 1H, H-6); 8.46 (d, 1H, H-9); 8.57 (dd,
1H, H-7); 3.91 (s, 3H, NCH3); 2.54 (s, 3H, CCH3). Anal.
(C13H10N7OCl) C, H, N.
3-((1,2,4-Oxa d ia zol-3-m et h yl)-5-yl)-8-ch lor op yr a zolo-
[5,1-c][1,2,4]ben zotr ia zin e 5-Oxid e, 5b. A suspension of 2b
(0.45 mmol, 150 mg) and NH2OH hydrochloride (0.66 mmol,
50 mg) in dioxane (2.0 mL) was added to AcOH (2.0 mL) and
a 10% aqueous solution of NaOH (0.4 mL). The suspension
was kept at 100 °C for 5 h and monitored by TLC (CH2Cl2/
MeOH, 10:0.5 v/v, as eluent). The final precipitate was filtered
and purified by recrystallization: yellow crystals. IR ν cm-1
:
1600, 1550. 1H NMR (CDCl3) δ: 8.65 (s, 1H, H-2); 8.50 (d, 1H,
H-6); 8.48 (d, 1H, H-9); 7.70 (dd, 1H, H-7); 2.50 (s, 3H, CH3).
Anal. (C12H7N6O2Cl) C, H, N.
3-(N-F or m ylca r ba m oyl)-8-ch lor op yr a zolo[5,1-c][1,2,4]-
ben zotr ia zin e 5-Oxid e, 6. From compound 2a (0.47 mmol,
150 mg) by treatment with H2O/AcOH at 70-80 °C. On cooling
the precipitate of the desired compound was formed: yellow
crystals. IR ν cm-1: 3220, 1730, 1680, 1560. 1H NMR (DMSO-
d6) δ: 11.24 (bs, 1H, NH, exchang.); 9.25 (s, 1H, CHO); 8.92
(s, 1H, H-2); 8.50 (m, 2H, H-6, H-9); 7.88 (dd, 1H, H-7). Anal.
(C11H6N5O3Cl) C, H, N.
Gen er a l P r oced u r e for Syn th esis of 2a a n d 2b. To a
suspension of compound 11 (1.9 mmol, 500 mg) in anhydrous
toluene (10 mL) and anhydrous DMF (1 mL) was added
dimethylformamide-dimethyl acetal (DMF-DMA) or di-
methylacetamide-dimethyl acetal (DMA-DMA) (6.8 mmol)
to obtain the intermediate 2a or 2b, respectively. The reaction
was kept at 100-110 °C and was monitored by TLC (toluene/
EtOAc/AcOH, 8:2:1 v/v/v, as eluent). The obtained residue was
filtered and washed by diethyl ether.
3-(N-(Hyd r oxyla m in om eth ylen e)ca r ba m oyl)-8-ch lor o-
p yr a zolo[5,1-c][1,2,4]ben zotr ia zin e 5-Oxid e, 7. Compound
2a (0.33 mmol, 100 mg) and NH2OH hydrochloride (0.46 mmol,
30 mg) in dioxane (1.0 mL) were added to AcOH (1.0 mL) and
a 10% aqueous solution of NaOH (0.2 mL). The suspension
was kept at room temperature for 2 h, and the final precipitate
was filtered and recrystallized: yellow crystals. IR ν cm-1
:
3320-3140, 1670, 1610, 1560. 1H NMR (DMSO-d6) δ: 11.04
(s, 1H, OH, exchang.); 10.00 (d, 1H, NH, exchang.); 8.82 (s,
1H, H-2); 8.52 (m, 2H, H-6, H-9); 7.91 (dd, 1H, H-7); 7.78 (d,
1H, CH, exchang.). Anal. (C11H7N6O3Cl) C, H, N.
3-(N-(Dim eth yla m in om eth ylen e)ca r ba m oyl)-8-ch lor o-
p yr a zolo[5,1-c][1,2,4]ben zotr ia zin e 5-Oxid e, 2a . From 1
1
with DMF-DMA; yellow crystals. IR ν cm-1: 1640, 1560. H
3-((ter t-Bu t oxyca r b a m oyl)a m in o)-8-ch lor op yr a zolo-
[5,1-c][1,2,4]ben zotr ia zin e 5-Oxid e, 9. Compound 81 (0.19
mmol, 50 mg) was suspended with NEt3 and diphenyl phosphor-
azidate (DPPA), in equimolar amount, in tert-butyl alcohol (5
mL). The reaction was kept at refluxing temperature for 8 h,
monitored by TLC (toluene/EtOAc/AcOH, 8:2:1 v/v/v, as elu-
ent), and the final solution was evaporated to dryness obtain-
ing a residue that was purified by recrystallization: red
crystals. IR ν cm-1: 3360, 1690, 1520. 1H NMR (CDCl3) δ: 8.56
(s, 1H, H-2); 8.43 (d, 1H, H-6); 8.30 (d, 1H, H-9); 7.51 (dd, 1H,
H-7); 6.85 (bs, 1H, NH, exchang.); 1.50 (s, 9H, (CH3)3). Anal.
(C14H14N5O3Cl) C, H, N.
NMR (DMSO-d6) δ: 8.68 (s, 1H, CH); 8.64 (s, 1H, H-2); 8.46
(m, 2H, H-6, H-9); 7.82 (dd, 1H, H-7); 3.21 (s, 3H, N-CH3);
3.19 (s, 3H, NCH3). Anal. (C13H11N6O2Cl) C, H, N.
3-(N-(1-Dim eth yla m in oeth ylid en e)ca r ba m oyl)-8-ch lo-
r op yr a zolo[5,1-c][1,2,4]ben zotr ia zin e 5-Oxid e, 2b. From
1 with DMA-DMA; yellow crystals. IR ν cm-1: 1610, 1550.
1H NMR (CDCl3) δ: 8.56 (s, 1H, H-2); 8.48 (d, 1H, H-6); 8.42
(d, 1H, H-9); 7.58 (dd, 1H, H-7); 3.29 (s, 3H, N-CH3); 3.18 (s,
3H, NCH3); 2.41 (s, 3H, CCH3). Anal. (C14H13N6O2Cl) C, H, N.
Gen er a l P r oced u r e for Syn th esis of 3a a n d 3b. To a
suspension of intermediate 2a or 2b (0.13 mmol) in AcOH (3

2224 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costanzo et al.
3-Am in o-8-ch lor op yr a zolo[5,1-c][1,2,4]b en zot r ia zin e
5-Oxid e, 10. Compound 9 (0.15 mmol, 50 mg) was hydrolyzed
in a solution of 16 M HCl (1.0 mL) and concentrated AcOH
(1.5 mL) for 1 h at refluxing temperature. The reaction was
monitored by TLC (toluene/EtOAc/AcOH, 8:2:1 v/v/v, as elu-
ent). The residue obtained by alkalinization with a 10%
aqueous solution of NaOH was filtered and purified: red
crystals. IR ν cm-1: 3440-3360, 1570. 1H NMR (CDCl3) δ: 8.41
(d, 1H, H-6); 8.19 (d, 1H, H-9); 7.73 (s, 1H, H-2); 7.45 (dd, 1H,
H-7); 3.66 (bs, 2H, NH2, exchang.). Anal. (C9H6N5OCl) C, H,
N.
1H, H-4′); 7.40 (s, 1H, H-3); 5.80 (bs, 2H, NH2 exch.); 3.70 (s,
2H, CH2). Anal. (C11H8N5O2Cl) C, H, N.
1-(2-Nit r o-5-ch lor op h en yl)-5-a m in op yr a zole-4-a cet -
a m id e, 15. From 14 by treatment with concentrated H2SO4
at 60 °C. The end of the reaction was monitored by TLC
(CHCl3/MeOH, 10:1 v/v, as eluent). The final solution was
added to ice-H2O and made alkaline with aqueous NH3. The
obtained precipitate was filtered and recrystallized with
H2O: yellow crystals, yield 60%; mp 181-182 °C. IR ν cm-1
:
3400-3200. 1H NMR (DMSO-d6) δ: 8.05 (d, 1H, H-3′); 7.80
(d, 1H, H-6′); 7.70 (dd, 1H, H-4′); 7.25 (s, 1H, H-3); 7.20 (bs,
1H, NH exch.); 6.90 (bs, 1H, NH exch.); 5.40 (s, 2H, NH2 exch.);
3.10 (s, 2H, CH2). Anal. (C11H10N5O3Cl) C, H, N.
3-(P yr r ol-1-yl)-8-ch lor op yr a zolo[5,1-c][1,2,4]ben zotr i-
a zin e 5-Oxid e, 11. A suspension of compound 10 (0.42 mmol,
100 mg) in dimethoxytetrahydrofurane (0.42 mmol, 0.05 mL)
and AcOH (6.0 mL) was refluxed for 2 h, following a described
procedure.21 The obtained solution was then evaporated to
3-(Ca r b a m oylm et h yl)-8-ch lor op yr a zolo[5,1-c][1,2,4]-
ben zotr ia zin e 5-Oxid e, 16. From 15 by treatment with a
10% aqueous solution of NaOH and few milliliters of diethyl-
ene glycol dimethyl ether (diglyme) at room temperature. The
final precipitate was filtered and purified: light-yellow crys-
tals. IR ν cm-1: 3400-3120, 1570. 1H NMR (DMSO-d6) δ: 8.45
(d, 1H, H-6); 8.35 (d, 1H, H-9); 8.25 (s, 1H, H-2); 7.75 (dd, 1H,
H-7); 7.45 (bs, 1H, NH exch.); 7.05 (bs, 1H, NH exch.); 3.55 (s,
2H, CH2). Anal. (C11H8N5O2Cl) C, H, N.
3-Ca r bom eth yl-8-ch lor op yr a zolo[5,1-c][1,2,4]ben zotr i-
a zin e 5-Oxid e, 17. From 16 by treatment with concentrated
H2SO4/NaNO2 as previously described:3 dark-yellow crystals.
IR ν cm-1: 3300-2800, 1570. 1H NMR (DMSO-d6) δ: 12.6 (bs,
1H, OH exch.); 8.42 (d, 1H, H-6); 8.34 (d, 1H, H-9); 8.26 (s,
1H, H-2); 7.74 (dd, 1H, H-7); 3.80 (s, 2H, CH2). Anal. (C11H7N4O3-
Cl) C, H, N.
dryness and the residue purified: red crystals. IR ν cm-1
:
1570. 1H NMR (DMSO-d6) δ: 8.72 (s, 1H, H-2); 8.42 (d, 1H,
H-6); 8.35 (d, 1H, H-9); 7.76 (dd, 1H, H-7); 7.45 (m, 2H, H-3′,
H-4′ pyrr.); 6.34 (m, 2H, H-2′, H-5′ pyrr.). Anal. (C13H8N5OCl)
C, H, N.
Gen er a l P r oced u r e for Syn th esis of 12c a n d 12d . The
suitable 3-oxopropanenitrile (c,d ) was reacted with 5-chloro-
2-nitrophenylhydrazine following a reported method.4
1-(2-Nitr o-5-ch lor op h en yl)-4-(th ien -2-yl)-5-a m in op yr -
a zole, 12c. From oxopropanenitrile c; reaction run under
nitrogen; orange crystals, yield 35%; mp 126-127 °C after
1
recrystallization from EtOH/H2O. IR ν cm-1: 3420-3280. H
NMR (CDCl3) δ: 7.98 (d, 1H, H-3′); 7.70 (m, 2H, H-6′, H-3);
7.58 (dd, 1H, H-4′); 7.26 (m, 1H, H-4′′ 4-thienyl); 7.08 (m, 2H,
H-2′′, H-3′′ 4-thienyl); 3.90 (bs, 2H, NH2, exchang.). Anal.
(C13H9N4O2SCl) C, H, N.
3-(1-Oxo-2-(d im et h yla m in o)vin yl)-8-ch lor op yr a zolo-
[5,1-c][1,2,4]ben zotr iazin e 5-Oxide, 18. Following the method
described in the literature,13 anhydrous DMF (3.88 mmol,
0.301 mL) was added dropwise, with vigorous stirring, to
POCl3 (3.24 mmol, 0.3 mL), mantaining the temperature at
maximum 30 °C. The mixture was stirring for 5 min; then
compound 17 (1.08 mmol, 300 mg) solubilized in anhydrous
DMF was added. The resulting solution was stirred at 70 °C
for 2 h and then was poured on ice and neutralized by the
addition of anhydrous K2CO3. The final suspension was made
strongly alkaline with a 40% aqueous solution of NaOH,
mantaining the temperature at 50 °C to completely eliminate
the NHMe2 which is formed during the reaction.The final
precipitate was filtered and washed well with H2O. The
obtained product was enough pure for the next step: dark-
1-(2-Nitr o-5-ch lor op h en yl)-4-(th ien -3-yl)-5-a m in op yr -
a zole, 12d . From oxopropanenitrile d ; orange crystals, yield
40%; mp 133-135 °C after recrystallization from ethanol/
water. IR ν cm-1: 3460-3180. 1H NMR (CDCl3) δ: 7.95 (d,
1H, H-3′); 7.70 (d, 1H, H-6′); 7.65 (s, 1H, H-3); 7.55 (dd, 1H,
H-4′); 7.43 (dd, 1H, H-4′′ 4-thienyl); 7.22 (m, 2H, H-2′′, H-5′′
4-thienyl); 3.80 (bs, 2H, NH2, exchang.). Anal. (C13H9N4O2SCl)
C, H, N.
Gen er a l P r oced u r e for Syn th esis of 13c a n d 13d . The
cyclization to pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide system
was obtained from the suitable 5-aminopyrazoles 12c and 12d
in alkaline medium, according to a previously reported
method.1-4
1
red crystals. IR ν cm-1: 1650, 1570. H NMR (CDCl3) δ: 9.14
3-(Th ien -2-yl)-8-ch lor op yr a zolo[5,1-c][1,2,4]b en zot r i-
a zin e 5-Oxid e, 13c. From 12c; red crystals. IR ν cm-1: 1570.
1H NMR (CDCl3) δ: 8.48 (d, 1H, H-6); 8.36 (d, 1H, H-9); 8.28
(s, 1H, H-2); 7.62 (dd, 1H, H-4′ 3-thienyl); 7.56 (dd, 1H, H-7);
7.34 (dd, 1H, H-2′ 3-thienyl); 7.12 (dd, 1H, H-3′ 3-thienyl).
Anal. (C13H7N4OClS) C, H, N.
3-(Th ien -3-yl)-8-ch lor op yr a zolo[5,1-c][1,2,4]b en zot r i-
a zin e 5-Oxid e, 13d . From 12d ; red crystals. IR ν cm-1: 1570;
1H NMR (CDCl3) δ: 8.48 (d, 1H, H-6); 8.38 (d, 1H, H-9); 8.32
(s, 1H, H-2); 7.88 (dd, 1H, H-2′ 3-thienyl); 7.62 (dd, 1H, H-5′
3-thienyl); 7.54 (dd, 1H, H-7); 7.42 (dd, 1H, H-4′ 3-thienyl).
Anal. (C13H7N4OClS) C, H, N.
3-(Th ien -2-yl)-8-ch lor op yr a zolo[5,1-c][1,2,4]b en zot r i-
a zin e, 13c′. From 13c by treatment with triethyl phosphite
according a previously cited method;3 light-red crystals. IR ν
cm-1: 1610. 1H NMR (CDCl3) δ: 8.56 (d, 1H, H-6); 8.44 (m,
2H, H-2 and H-9); 7.92 (dd, 1H, H-4′ 3-thienyl); 7.70 (dd, 1H,
H-7); 7.44 (dd, 1H, H-2′ 3-thienyl); 7.22 (dd, 1H, H-3′ 3-thienyl).
Anal. (C13H7N4OClS) C, H, N.
(s, 1H, CHO); 8.43 (d, 1H, H-6); 8.35 (d, 1H, H-9); 8.10 (s, 1H,
H-2); 7.54 (dd, 1H, H-7); 7.05 (s, 1H, CHNMe2); 2.98 (s, 6H,
NMe2). Anal. (C13H12N5O2Cl) C, H, N.
3-(1-Oxo-2-h yd r oxyvin yl)-8-ch lor op yr a zolo[5,1-c][1,2,4]-
ben zotr ia zin e 5-Oxid e, 19. Compound 18 (1.1 mmol, 350 mg)
was suspended in 2.5 mL of diglyme and 2.0 mL of a 20%
aqueous solution of NaOH, at 50 °C. After 2 h the starting
product was completely hydrolyzed. The suspension was cooled
and acified with 6 M HCl obtaining a red precipitate that was
purified by recrystallization: red crystals. IR ν cm-1: 1570.
1H NMR (CDCl3) δ: 9.20 (bs, 1H, CHOH); 8.88 (bs, 1H, H-2);
8.42 (m, 2H, H-6 and H-9); 7.58 (dd, 1H, H-7); 7.20 (d, 1H,
CHOH); after exchange with D2O the spectra appeared; 9.40
(s, 1H, CHO); 9.20 (s, 1H, CHOH); 8.88 (s, 1H, H-2); 8.42 (m,
1
2H, H-6 and H-9); 7.58 (dd, 1H, H-7). H NMR (DMSO-d6) δ:
8.70 (bs, 2H, CHO, CHOH); 8.42 (d, 1H, H-6); 8.36 (d, 1H, H-9);
8.26 (bs, H, H-2); 7.76 (dd, 1H, H-7). Anal. (C13H7N4O2Cl) C,
H, N.
3-(Isoxa zol-4-yl)-8-ch lor opyr a zolo[5,1-c][1,2,4]ben zotr i-
a zin e 5-Oxid e, 20. Compound 19 (0.180 mmol, 50 mg) was
suspended in EtOH, and NH2OH hydrochloride was added and
maintained at refluxing temperature. The reaction was moni-
tored by TLC (CHCl3/MeOH, 10:1 v/v, as eluent), and after 3
h the final product was completely formed. The solvent was
removed under reduced pressure, and the red residue was
purified: red crystals. IR ν cm-1: 1570. 1H NMR (CDCl3) δ:
9.20 (s, 1H, H-5 isox); 8.80 (s, 1H, H-3 isox); 8.50 (d, 1H, H-6);
8.38 (d, 1H, H-9); 8.26 (s, 1H, H-2); 7.60 (dd, 1H, H-7). Anal.
(C12H6N5O2Cl) C, H, N.
1-(2-Nit r o-5-ch lor op h en yl)-5-a m in op yr a zole-4-a cet o-
n itr ile, 14. The 2-nitro-5-chlorophenylhydrazine1 (5.34 mmol,
1 g) was reacted with 2-oxosuccinonitrile (8.01 mmol, 865 mg)
in EtOH at refluxing temperature. The reaction was monitored
by TLC (toluene/EtOAc, 8:2 v/v, as eluent) until the starting
material disappeared.The final solution was evaporated to
dryness, and the final residue was purified by recrystallization
with an 80% aqueous solution of EtOH: yellow crystals, yield
1
52%; mp 183-184 °C. IR ν cm-1: 3400-3340, 2240. H NMR
(DMSO-d6) δ: 8.10 (d, 1H, H-3′); 7.85 (d, 1H, H-6′); 7.75 (dd,

Benzodiazepine Receptor Ligands
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2225
Ra d ioliga n d Bin d in g Assa y. [3H]Ro15-1788 (s.a. 32.2
Ci/m mol-1) was purchased from DuPont New England Nuclear
(Boston, MA). All other reagents were obtained from com-
mercial suppliers.
experimental room was slightly illuminated. Mice were treated
po with either drugs or CMC and 30 min later individually
placed into the center of the lighted side of the box, facing away
from the divider. Behavior was observed for 5 min, and time
spent in the illuminated side of the box and the number of
crosses from one side to the other were measured. A crossover
was counted when all four paws were in the opposite side of
the box. Statistical analysis was performed by means of
Student’s t-test.
Bovine cerebral cortex membranes were prepared as previ-
ously described.22 In brief, tissue was homogenized in10
volumes of ice-cold 0.32 M sucrose containing protease inhibi-
tors. The homogenate was centrifuged for 5 min at 2000g at 4
°C, and the suspension was recentrifuged for 30 min at 30000g
at 4 °C. The resulting pellet was resuspended in 50 mM Tris-
citrate buffer at pH 7.4 (buffer T), homogenized, and centri-
fuged for 30 min at 30000g at 4 °C. The resulting pellet was
subjected to washing procedures to remove endogenous GABA.23
For this purpose the membranes were frozen, thawed, resus-
pended in 32 volumes of T buffer, containing 0.01% Triton
X-100 and protease inhibitors, homogenized, incubated for 60
min at 37 °C, and centrifuged for 30 min at 30000g at 4 °C.
The pellet was resuspended in buffer T, homogenized, and
recentrifuged. The washing procedure was repeated three
times. The washed membranes were incubated with [3H]Ro15-
1788 (∼0.2 nM, Kd ) 0.60 nM) for 90 min at 0 °C in 500 µL of
buffer T. The incubation was terminated by dilution to 5 mL
with ice-cold buffer T, followed immediately by rapid filtration
through glass fiber Whatman GF/C filters. The filters were
then washed (2 × 5 mL) with buffer T, and the amount of
radioactivity retained on the filters was determinated by
Packard 1600 TR liquid scintillation counter at 66% efficiency.
Nonspecific binding was estimated in the presence of 10 µM
diazepam. The compounds were dissolved in EtOH (or DMSO)
and tested at a concentration of 10 µM. For the active
compounds, the IC50 values were determined and Ki values
were derived according to the equation of Cheng and Pursoff.24
Protein concentration was assayed by the method of Lowry et
al.25 IC50 determination for GABA ratio values was carried out
in the absence and presence of 10 µM GABA.
P h a r m a cologica l Meth od s. Male Swiss-Webster mice
weighing 22-26 g (Harlan Nossan) were used. Twelve mice
were housed per cage. The cages were taken into the experi-
mental room 24 h before testing and left under a normal 12-h
light/dark cycle (lights on 7:00 h). The animals were fed a
standard laboratory diet and tap water ad libitum. All experi-
ments were conducted between 9:00 and 17:00.
Dr u gs: The following commercial drugs were used diazepam
(valium 10, Roche), flumazenil (Ro15-1788, Roche), and pen-
tylenetetrazole (Sigma). Drug concentrations were prepared
in such a way that the necessary dose could be injected in a
10 mL/kg volume of carboxymethylcellulose (CMC), 1%.
Rota -r od test:26 The apparatus consisted of a base platform
and a rotating rod of 3-cm diameter with a nonslippery surface.
This rod was placed at a height of 15 cm from the base. The
integrity of motor coordination was assessed at a rotating
speed of 24 rpm, counting the number of falls from the rod in
30 s, 25 min after treatment. The statistical analysis was
performed by means of the Student’s t-test.
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