Novel 3-aroylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted, ligands at GABAA/benzodiazepine receptor complex: Synthesis, pharmacological and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2008.02.058

The synthesis and binding studies of a series of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted are reported. High-affinity ligands at benzodiazepine site on GABA_A receptor complex (GABA_A/BzR complex) were obtained when the 3-aroyl substituent is represented by a five-member heteroaroyl ring (furoyl-, thenoyl-, and pyrroyl-). Moreover the type of heteroaroyl ring at position 3 influences the feature of the substituent at position 8 to obtain high-affinity ligands: a 'hydrogen-bond acceptor ring' at position 3 is synergic with an electron donor substituent at position 8, while a 'hydrogen-bond donor ring' is synergic with a withdrawing substituent. Compounds 8a, 9b, and 11 were deeply studied in vivo for their pharmacological effects considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motor activity and explorative activity, anxiolytic-like effects, mouse learning and memory modulation, and ethanol-potentiating action. To rationalize and qualitatively interpret the GABA_A/Bz binding affinities of compounds 8a and 11, a dynamic molecular modeling study has been performed, with the aim of assessing the preferred geometry of protein-ligand complex.

Full text of DOI:10.1016/j.bmc.2008.02.058

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4471–4489
Novel 3-aroylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides
8-substituted, ligands at GABAA/benzodiazepine receptor complex:
Synthesis, pharmacological and molecular modeling studies
Gabriella Guerrini,a,* Giovanna Ciciani,a Giovanni Cambi,a Fabrizio Bruni,a
Silvia Selleri,a Fabrizio Melani,b Marina Montali,c Claudia Martini,c
Carla Ghelardini,d Monica Norcinid and Annarella Costanzoa
aDipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente
`
attivi (HeteroBioLab) Universita degli Studi di Firenze, Via U. Schiff, 6, 50019 Polo Scientifico, Sesto Fiorentino (Firenze), Italy
bDipartimento di Scienze Farmaceutiche, Laboratorio di Molecular Modeling, Cheminformatics and QSAR
`
Universita degli Studi di Firenze, Via U. Schiff, 6, 50019 Polo Scientifico, Sesto Fiorentino (Firenze), Italy
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita degli Studi di Pisa,
c
`
via Bonanno, 6, 56126 Pisa, Italy
Dipartimento di Farmacologia Preclinica e Clinica Aiazzi-Mancini, Universita degli Studi di Firenze,
Viale Pieraccini, 6, 50139 Firenze, Italy
d
`
Received 19 October 2007; revised 13 February 2008; accepted 19 February 2008
Available online 5 March 2008
Abstract—The synthesis and binding studies of a series of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted are
reported. High-affinity ligands at benzodiazepine site on GABAA receptor complex (GABAA/BzR complex) were obtained when
the 3-aroyl substituent is represented by a five-member heteroaroyl ring (furoyl-, thenoyl-, and pyrroyl-). Moreover the type of het-
eroaroyl ring at position 3 influences the feature of the substituent at position 8 to obtain high-affinity ligands: a ‘hydrogen-bond
acceptor ring’ at position 3 is synergic with an electron donor substituent at position 8, while a ‘hydrogen-bond donor ring’ is syn-
ergic with a withdrawing substituent. Compounds 8a, 9b, and 11 were deeply studied in vivo for their pharmacological effects con-
sidering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motor activity and
explorative activity, anxiolytic-like effects, mouse learning and memory modulation, and ethanol-potentiating action. To rationalize
and qualitatively interpret the GABAA/Bz binding affinities of compounds 8a and 11, a dynamic molecular modeling study has been
performed, with the aim of assessing the preferred geometry of protein–ligand complex.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
barbiturates, and anesthetics. GABAA receptor is a het-
ero-oligomeric complex composed of five transmem-
Gamma-aminobutyric acid (gaba), the major inhibitory
neurotransmitter in mammalian brain, targets the iono-
phore GABAA and GABAC receptors and the metabo-
tropic GABAB receptors. Of these, it is the GABAA
receptor family which has been the most widely studied
since this family is the site of action of a number of clin-
ically important drugs, including benzodiazepines (BZs),
brane spanning subunits assembled to form
a
pentamer: there are 16 different subunits (a1–6, b1–3,
c1–3, d, e, p, h) or 19 if the so-called GABAC subunits
are included (q1–3). The majority of GABAA receptor
subtypes contain a-, b-, and c-subunits arranged in a
2:2:1 stoichiometry and concatenated as cbaba, counter-
clock-wise from synaptic cleft.1 The Bzs binding occurs
at the interface of a and c subunits and the major BZ-
sensitive GABAA receptors in the brain are a1b2c2,
a2b3c2, a3b3c2, a5b3c2. These show distinct regional
distribution in the brain which provokes the question
of whether GABAA receptor subtypes are linked to dis-
tinct pharmacological functions.2–4 Currently, the genet-
Keywords: GABAA/benzodiazepine receptor ligands; Tricyclic hetero-
aromatic system; 3-Acyl derivatives pyrazolo[5,1-c][1,2,4]benzotriazine
5-oxides.
*
Corresponding authors. Tel.: +39 055 4573766; fax +39 055
4573671; e-mail: gabriella.guerrini@unifi.it
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.058

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G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
ic approach (the use of transgenic mice with point muta-
tion on a-subunit) and the development of ligands en-
dowed with selective efficacy have permitted the
obtainment of data suggesting that a certain subtype
receptor might be responsible for mediating specific
pharmacological effects of BZs.2,5–8
via c subunit from the N-terminal region of transmem-
brane domain M1 through M2 and the proximal portion
of the M2–M3 loop. The benzodiazepine binding ‘pro-
duces’, at the end, a decreased energy requirement for
channel opening.
To obtain drugs which are more selective than BZs, the
development of GABAA receptor subtype selective li-
gands or functionally selective ligands is the current
strategy of medicinal chemistry researchers. With this
aim in mind, we have identified in a previously synthe-
sized compound, the 3-(3-thienyl)-8-chloropyrazol-
o[5,1-c][1,2,4]benzotriazine 5-oxide,19 the lead for our
new research. This compound is a selective agonist en-
dowed with anticonvulsant activity, acting trough the
GABAA/BZs receptor complex, since its effect were
antagonized by flumazenil. Starting from this compound
a chemical modification at position 3 was made obtain-
ing new 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide 8-derivatives. These compounds can be considered
metabolically more stable than ester derivatives previ-
ously studied20–22 and strictly related to Indiplon23,24
and Ocinaplon,25 two heteroarylmethanones endowed
with selective agonist profile (respectively, sedative and
anxiolytic activity) through the GABAA/benzodiazepine
receptor complex.9,10,26–28 These compounds belong to
pyrazolo[1,5-a]pyrimidines chemical family and the 3-
acylpyrazolopyrimidine core is easily recovered in our
pyrazolobenzotriazine system (see Chart 1). This modi-
All data are consistent with an important role of a1-con-
taining GABAA receptors in mediating sedative effects
and part of anticonvulsant activity and amnesic action;
a2- and/or a3-containing GABAA receptors in mediat-
ing the anxiolytic-like action of benzodiazepines; a5-
containing GABAA receptors are important for the
cognitive effects and/or in the neurobehavioral action
of alcohol.6,9–15
The mechanism by which allosteric modulators, as ben-
zodiazepines, influence the GABAA receptor currents is
unclear. Recently, the conformational rearrangements
triggered by benzodiazepines binding in or near the
GABA binding site (considering that GABA binding
pocket is physically linked to the Bzs binding site) have
been reported.16–18 It seems that GABAA/BzR positive
modulators can induce change at a specific region
formed by 11 amino acids on GABA binding site
(aMet113-aLeu132 or Loop E) and this could elicit, at
18
the end, different functional effects.16,17 Other model
provided evidence that the transduction of signal, after
benzodiazepine binding at the a/c interface, happens
LEAD
Ki = 36.3 nM
Anticonvulsant
N
N
Cl
S
N
N+
O-
N
N
R8
R/Ar
O
N
N+
O-
O
H3C
CH3
N
N
N
N
N
N
N
O
N
S
O
N
Ocinaplon25
Indiplon 23, 24
Chart 1.

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4473
3-Carbonylderivatives:
R/Ar =
N
O
H
1
2
3
4
5
6
N
Cl
CH3
R/Ar
Ph
IV, V, IX
N
N
Method A
N+
O-
o-OCH3Ph
p-OCH3Ph
3-pyridil
N
O
N
Cl
N
Cl
N
Ar
N+
O-
1,3-benzodioxol-5-yl 7
NH2
Method B or
Method C
Method D
NO2
NH
NH
2-furyl
8
2-thienyl
3-thienyl
2-pirrolyl
9
I
O
N
N
10
11
O
Cl
Cl
MeI/
DMF
R/Ar
1-methylpirrol-2-yl 12
1-methylpirrol-3-yl 13
OH
N
NO2
N
N+
II
III, VI, VII, VIII
O-
Scheme 1. Reagents: Method A: I30 DMF/POCl3 for compound 1. Method B: II30 (i) thionyl chloride; (ii) ArH, SnCl2 for compounds 5, 8–9, 11–13;
12 was also obtained by the alkylation of 11. Method C: II30 (i) thionyl chloride; (ii) phenylboronic acid, Cs2CO3 for compound 3. Method D: (i)
NaOH 10% solution, 50–60 ꢁC for compounds 2, 4, 6–10 (respectively, from starting material: III, IV, V, VI, VII, VIII and IX). For compounds 8
and 9 method B and D were indifferently used (yields shown on Supplementary data, Table 1).
fication at position 3 permits us to further explore the
type of interaction that exists between the substituent
at position 3 and the receptor protein. Moreover to de-
fine the role of 5-oxide group,22,29 also in the new 3-ar-
oyl derivatives series, we have provided for the synthesis
of some 5-deoxy derivatives.
For the latter method (method D for compounds 2, 4, 6–
10), which gives the 3-acylpyrazolobenzotriazine system
by ring closure in alkaline medium,30 the synthesis of
the starting compounds III–IX was developed as shown
in Scheme 2. In particular the 5-chloro-2-nitro-
phenylhydrazine31 was reacted with 2-ethoxymethylen-
3-oxobutanenitrile32 or with 2-(dimethylaminomethy-
len)-3-oxo-3-arylpropanenitrile33–36 and the intermedi-
ate hydrazinyl derivatives III, VI, VII, VIII or the
5-aminopyrazoles IV, V, IX were obtained (Scheme 2)
and used in the next cyclization in 10% sodium hydrox-
ide solution.
1.1. Chemistry
Two synthetic approaches to obtain the desired 3-acyl-8-
chloropyrazolo[5,1-c] [1,2,4]benzotriazine 5-oxides 1–13
were developed consisting in: modification of position
3 (e.g., –H, –COOH substitution), by functionalization
of this position with the keto group at a later stage of
the tricyclic system synthesis (method A, B, C in Scheme
1); intramolecular cyclization under basic conditions of
the 5-aminopyrazole or hydrazinyl derivative, bearing
at 4-position the suitable keto group (method D in
Scheme 1).
A particular reactivity and regiospecificity depending on
the type of acid catalyst used was observed when the 2-
ethoxymethylen-3-oxobutanenitrile32 was reacted with
5-chloro-2-nitrophenylhydrazine. In fact when the reac-
tion was carried out in ethanol/acetic acid (Scheme 2)
the ring closure proceeded involving, as the first step,
the primary amino group of hydrazine giving the en-
hydrazinyl derivative III.
In the first approach (method A), the starting material 8-
chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide30
I
was reacted according to the Vilsmayer conditions
(DMF/POCl3) and the desired 3-formyl derivative 1, to-
gether with the corresponding 5-deoxide 1R, was ob-
tained after chromatographic separation (10/1 ratio).
In the attempt to obtain the 5-aminopyrazole-4-etha-
none derivative, starting products (2-ethoxymethylen-
3-oxobutanenitrile32 and 5-chloro-2-nitrophenylhydr-
azine) were reacted using HCl concd as catalyst
(Scheme 2). Also in this case the first intermediate
was the en-hydrazinyl derivative III (not isolated and
detected by TLC) which gave a regiospecific ring clo-
sure involving the –COCH3 group instead of the –CN
group and the final compound was the 1-(2-nitro-5-
chlorophenyl)-5-methylpyrazole-4-carbonitrile, III0. It
is important to note that this type of ring closure has
occurred only for intermediate III in HCl concd condi-
tions: the other hydrazinyl derivatives (VI, VII, and
VIII) in the same condition never gave the correspond-
When the starting material was the 3-carboxy-8-chloro-
pyrazolo[5,1-c][1,2,4] benzotriazine 5-oxide30 II, the
treatment with thionyl chloride gave the corresponding
3-acylchloride as intermediate which, exploiting the Fri-
edel–Craft reaction, was directly converted into final
products, 5, 8–9, 11–13 (method B). Alternatively when
the 3-acylchloride was reacted with phenyl boronic acid,
(method C), in Suzuki coupling conditions compound 3
was obtained in good yield.

4474
G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
NH2
R'
CN
NH
Cl
R/Ar
H
O
+
NO2
i
i
O
N
NH
NH
O
N
Cl
Cl
Ar
R/Ar
NH2
N
NO2
NO2
R/Ar= CH3
III
VI
VII
VIII
Ar= o-OCH3Ph
3-Py
IV
V
IX
1,3-benzodioxol-5-yl
2-furyl
2-thienyl
3-tienyl
N
CN
N
ii
Cl
III
CH3
NO2
III’
Scheme 2. Reagents and conditions: (i) ethanol/acetic acid, reflux; (ii) ethanol/HCl concd, reflux R = Me, R0 = OEt.32 R = o-OCH3Ph, 1,3-
benzodioxol-5-yl, 3-Py, 2-furyl, 2-thienyl, 3-thienyl, R0 = NMe2.33–36
Nu:
N
a = EtO-
O
N
O
b = PhO-
N
Cl
c = PhCH2O-
R'
N
d = CH C-CH2O-
R/Ar
R/Ar
N
N+
O-
N
N+
O-
Compd
2a
2b
5a
6a
8a
8b
8c
8d
9a
R’
Compd
R/Ar
Me
p-OMePh
3-pyridyl
2-furyl
2-thienyl
3-thienyl
2-pyrrolyl
EtO
PhO
EtO
EtO
EtO
PhO
2
5
6
8
9
11
12
PhCH2O
CH C-CH2O
EtO
9b
9c
11a
11b
12a
12b
PhO
PhCH2O
EtO
PhO
EtO
PhO
Scheme 3. Reagents: 40% sodium hydroxide solution and ethanol for compounds 2a, 5a, 6a, 8a, 9a, 11a, and 12a; 40% sodium hydroxide solution,
tetrabutylammonium bromide NBu+Brꢀ, ROH, CH2Cl2 for compounds 2b, 8b, 9b, 11b, 8c, 9c, and 8d.

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4475
Table 1. BZR ligand affinity of new 3-acylpyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide
ing 4-carbonitrile derivatives, but evolve to 5-aminopy-
razoles as verified by previously laboratory experiences
(unpublished data).
N
O
N
R'
To evaluate the influence of the 8-substituent on bio-
logical activity, the aromatic nucleophilic substitution
of chlorine at position 8 was exploited, as depicted in
Scheme 3. To obtain compounds 2a, 5a, 6a, 8a, 9a,
11a, and 12a, starting materials 2, 5, 6, 8, 9, 11, and
12 were suspended in ethanol and 40% sodium hydrox-
ide solution as previously reported.29 Products 2b, 8b,
9b, 11b, 12b, 8c, 9c, and 8d were obtained by exploiting
the phase transfer catalyzed (PTC)37 chlorine displace-
ment at position 8 of the benzotriazine system. In this
procedure the suitable reagent (phenol, benzyl alcohol,
R
N
N+
X
Compound
R
R0
X
I%a or
Kib (nM)
1
H
Me
Cl
Cl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
—
—
64
2
551
902
53
or propargyl alcohol) is added to
a two-phase
2a
2b
3
Me
Me
OEt
OPh
Cl
system consisting of a strong aqueous sodium hydrox-
ide solution (40%), a catalyst (tetrabutylammonium
bromide, Bu4N+Brꢀ), and a methylene chloride solu-
tion of starting materials 2, 8, 9, 11, and 12 (see
Scheme 3).
Ph
3494
4200
1240
919
2400
2000
2900
72
4
o-OMe-Ph
p-OMe-Ph
p-OMe-Ph
3-Py
Cl
Cl
5
5a
6
OEt
Cl
6a
7
3-Py
1,3-Benzodioxol-5-yl Cl
OEt
To better explore the role of the N-oxide group, com-
pounds 9b and 11 were N5-deoxygenated, using tri-
ethylphosphite (TEP) as previously reported,38 yielding
compounds 9bR and 11R. All chemical data of com-
pounds described here are listed in Tables 1 and 2 avail-
able on ‘Supplementary data’.
8
2-Furyl
2-Furyl
Cl
OEt
8a
8b
8c
8d
9
28
2-Furyl
2-Furyl
2-Furyl
OPh
OCH2Ph
7
1.4
OCH2C@CH
Cl
3.5
62
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
3-Thienyl
2-Pyrrolyl
2-Pyrrolyl
2-Pyrrolyl
1.2. Binding studies
9a
9b
9c
10
11
11a
11b
12
12a
12b
13
9bR
11R
Dazc
Fluc
OEt
OPh
OCH2Ph
Cl
45
8.9
The GABAA/BzR complex binding affinity of new 3-
acylderivatives was evaluated by their ability to displace
[3H]flumazenil (Ro15-1788) from its specific binding in
bovine brain membrane. Binding data for new, synthe-
sized compounds (1–13, 2a, 5a, 6a, 8a, 9a, 11a, 12a,
2b, 8b, 9b, 11b, 12b, 8c, 9c, 8d, 9bR and 11R) and for ref-
erence compounds are reported in Table 1.
2.7
121
4
Cl
OEt
OPh
1836
276
5202
819
1300
380
14.7
1941
10
1-Methylpyrrol-2-yl Cl
1-Methylpyrrol-2-yl OEt
1-Methylpyrrol-2-yl OPh
1-Methylpyrrol-3-yl Cl
The following observations arise from the binding
studies’ results. First of all, the type of substituent
on the carbonyl group at position 3 resulted very
important. In the series of 8-chloroderivatives com-
pounds 1 and 2 (respectively, 3-formyl- and 3-acetyl
derivatives) have no or low-affinity value (1,
I% = 64%; 2, Ki = 551 nM) as compounds bearing at
position 3 a six-membered aroyl/heteroaroyl group
(3, 4, 5, 6 or the larger 7) which have a Ki in the range
of 4200–1240 nM.
2-Thienyl
2-Pyrrolyl
OPh
Cl
0.9
a Percent of inhibition of specific [3H]Ro15-1788 binding at 10 lM
concentration are means SEM of five determinations.
b Ki value are means SEM of five determinations.
c See Ref. 21.
of compound 10 with respect to the corresponding iso-
mer 3-(2-thenoyl)derivative 9 (10, Ki = 121 nM, 9,
Ki = 62 nM): this fact could be explained by the differ-
ent orientation of sulfur lone pairs that render the
interaction weaker.
When the substituent of the carbonyl group is a five-
membered heteroaroyl group (8, 9, 10 and 11) the
affinity strongly increases up to a Ki value of 4 nM
in the case of compound 11. Probably heteroaroyl
derivatives 8 (3-(2-furoyl)-, Ki = 72 nM) and 9, (3-
(2-thenoyl)-, 9, Ki = 62 nM), interact with receptor
protein in a different way with respect to 3-(pyrrol-2-
carbonyl)derivative 11 (Ki = 4.0 nM). In compounds
8 and 9 the lone pairs of oxygen and, to a lesser de-
gree, those of sulfur could be involved in a hydro-
gen-bond with the hydrogen-bond donor site (H1) on
the receptor protein. The presence of the 3-thenoyl
at position 3 causes a reduction of the affinity value
In the case of compound 11, which bears a 3-(pyrrol-2-
ylcarbonyl) moiety, the much higher potency
(Ki = 4.0 nM) could likely be due to a hydrogen-bond
interaction between the pyrrole-NH and the receptor
protein. The hypothesis that a hydrogen-bond acceptor
site (A2) may exist in the proximity of, or located at the
S1 site of BzR is yet suggested by Primofiore et al.39 On
the other hand, the importance of the pyrrole NH is

4476
G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
Table 2. Effects of the new compounds in comparison with diazepam on motor coordination and convulsion
Treatmenta
mg/kg po
Motor coordination rota-rod test
(16 rpm) falls in 3000
Anticonvulsant activity
n
Pretest
300
n
Shock latency (s)
Convulsion latency (s)
CMC 1%b
Diazepam
0.1 ml
1 (ip)
22
6
4.2 0.25
4.8 0.4
1.3 0.3
4.5 0.4*
38(7)c
48
273.3 11.6
No shocks***
522.25 60.35
No convulsions***
8a
9b
11
3
10
10
10
4.2 0.4
4.2 0.4
3.5 0.3
0.6 0.3
0.8 0.3
0.6 0.2
10(1)
10(1)
nd
nd
328.5 38.0*
488.2 26.0
10
30
3
10
10
10
3.8 0.5
3.8 0.3
3.6 0.5
0.8 1.1
1.6 0.8
2.0 0.8
13(8)
12(7)
16(5)
224.5 19.5
279.7 34.4
614.1 66.8***
460.3 68.9
542.6 69.3
766.6 92.6*
10
30
3
17
17
17
4.1 0.4
4.0 0.3
4.6 0.4
0.6 0.2
1.2 0.2
0.8 0.3
14(2)
14(2)
14(3)
297.5 17.2
264.7 17.5
329.6 31.5
498.8 64.2
647.8 144.8
535.2 90.5
10
30
a Treatment with new compounds was performed 30 min and diazepam (ip) 20 min before the test. In the rota-rod test the effect of treatments was
performed 15, 30, 45 and 60 min after the drug administration. The complete data are reported in Supplementary data (Table 4).
b Carboxymethylcellulose 1%.
c Number of dead mice.
* P < 0.05.
*** P < 0.001 versus control mice.
evidenced by the lack of affinity of the 3-(1-methylpyr-
rol-2-ylcarbonyl) derivative, 12, Ki = 5202 nM. The
shift of the N-methylpyrrole group from position 2-yl
to position 3-yl gave compound 13 with a better Ki va-
lue than compound 12 (13: Ki = 380 nM vs 12:
Ki = 5202 nM).
values were drastically reduced with respect to the 8-
chloro derivative 11 (11a, Ki = 1836 nM and 11b,
Ki = 276 vs 11, Ki = 4 nM). In this case we could assume
that the NH hydrogen bond with the receptor protein
‘shifts’ the whole molecule and this does not permit
the fitting of the 8-substituents, which are more awk-
ward than chloride (see Section 1.3).
When the 3-carbonylpyrazolobenzotriazine scaffold was
substituted at position 8 with groups such as -OEt,
(compounds 2a, 5a, 6a, 8a, and 9a) that previously
showed a general positive influence on affinity va-
lue,29,40 or –OPh (compounds 2b, 8b, and 9b), it was
observed that the order of substituent on binding was
OPh > OEt»Cl. The influence of 8-substitution is par-
ticularly evident if at position 3 is present a five-mem-
bered ring (furoyl- or thenoyl-): an increase of
affinity, with respect to 8 and 9, was shown (8a,
Ki = 28 nM and 8b, Ki = 7 nM vs 8, Ki = 72 nM; 9a,
Ki = 45 nM and 9b, Ki = 8.9 nM vs 9, Ki = 62 nM).
The importance of the methylene spacer, as in the case
of the 8-benzyloxy group, is evidenced in compounds 8c
and 9c which show an affinity value from 4- to 7-fold
higher compared to the corresponding 8-phenoxy deriv-
atives 8b and 9b (8c, Ki = 1.4 nM and 9c Ki = 2.7 nM vs
8b, Ki = 7 nM and 9b Ki = 8.9 nM). When the 3-(fur-2-
ylcarbonyl)pyrazolobenzotriazine scaffold was substi-
tuted at position 8 with a propargyloxy group (8d)
the binding-affinity value is in the same range of Ki
(3.5 nM) of 8c. This could probably be explained by
the large size of the lipophilic pocket in which the 8-
substituent fits, by an involvement of the ligand–
receptor protein p-systems and by a probable hydrogen
bond that involves the ethereal oxygen (see Section
1.3).
The different role of N5-oxide in these new derivatives is
evidenced in compounds 9bR and 11R. The lack of N5-
oxide group drastically reduces the affinity value only in
the case of compound 11 (11R, Ki = 1941 nM vs 11,
Ki = 4 nM). It appears that, in this case, the potential
three-centered hydrogen bond in which the 5-oxide
group is involved,19,38 is necessary to better anchor the
molecule to the receptor protein. In the case of com-
pound 9b the lack of the N5-oxide group maintains the
affinity value in the same value range (9bR Ki = 14.7 nM
vs 9b Ki = 8.9 nM), therefore the N-oxide group results
to be secondary to anchor the molecule on the receptor
protein.
1.3. Molecular modeling studies
In the present study we used the comparative (homol-
ogy) model developed by Ernst et al.41,42 to try to qual-
itatively interpret the GABAA/Bz binding affinities of
compounds 8a (Ki = 28 nM) and 11 (Ki = 4 nM), 3-
(fur-2-ylcarbonyl)- and 3-(1H-pyrrol-2-yl carbonyl)
derivatives, respectively. It could be hypothesized that
the different features of the heteroaroyl moiety present
in the two compounds, H-bond acceptor for 2-furoyl
and H-bond donor for 2-pyrroyl, are responsible for
the different way of interaction with receptor protein.
Surprisingly, when the 3-(1H-pyrrol-2-ylcarbonyl)pyra-
zolobenzotriazine scaffold is substituted at position 8
with a –OEt (11a), or a –OPh group (11b), the affinity
A dynamic molecular modeling study has been per-
formed with the aim of assessing the preferred geometry
of protein–ligand complex, starting from the likely con-

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4477
A
B
N
N
O
O
R'
N
R'
N
X
N
N
N+
O-
N+
O-
X
X
N
N
N
X
R'
R'
N
O
O
N
N+
O-
N
N+
O-
D
C
X = O
8a
11
R’ = OEt
X = NH
R’ = Cl
Figure 1. Four possible conformers (A–D) for each compound.
formations of the ligands. For each compound (8a and
11) four conformers A, B, C and D (Fig. 1) were selected
and ‘inserted’ in the binding site in four different modes
to obtain for each compound sixteen possible conforma-
tions (Fig. 2; for example, conformer A has been in-
serted in mode A1, A2, A3, and A4 and so on for
each conformer).
gen atom of the ethoxy group and the oxygen atom of
the furane ring and the receptor protein were selected.
These selected conformers showed the lowest conforma-
tional energy, that is 40 kcal/mol less with respect to the
mean value of all conformers collected during the MD
simulation. In particular, the oxygen atom of the furane
ring could engage a hydrogen bond either with hydroxyl
residue of cThr142 or the same residue on cThr81, while
the ethereal oxygen atom could engage the hydrogen
bond either with cArg144 (NH guanidine) or, alterna-
tively, with aHis101 (NH imidazole ring). The carbonyl
oxygen atom forms a strong hydrogen bond with
cArg132 (NH guanidine) and in part with the hydroxyl
group of cThr142. The N4-atom of the benzotriazine
system and the oxygen of the N-oxide group alterna-
tively interact with cArg132 (NH guanidine) and with
the hydroxyl group of aSer205; the same oxygen of
the N-oxide group in some conformations forms a
hydrogen bond with the hydroxyl group of aTyr209
(Fig. 3).
Ligands 8a and 11 were docked in the a1 (from Ile17 to
Gly223) and c2 (from Ile30 to Gly234) peptide chains
that were cut out from pentameric construct down-
loaded from Margot Ernst’s web page.43 The extracellu-
lar domain of the a1, b2, and c2 subunits is truncated N-
terminally, respectively, at Ile17, Val16, and Ile30 and
C-terminally at Gly223, Gly219, and Gly234. The bind-
ing site has been evidenced considering the most likely
residues that mediate the binding at GABAA/BzR com-
plex,44–50 and to construct the complex, a protein por-
˚
tion of 20 A radius has been used.
A molecular dynamic (MD) simulation of the complex
was carried out (see Section 3) and for each simulation
those conformers of ligand–protein complex that
showed an effective hydrogen-bond interaction were col-
lected. With the term ‘effective hydrogen-bond interac-
tion’ we mean those hydrogen bonds whose distance
Analogously, for compound 11 (Fig. 4) those conform-
ers of ligand–protein complex were selected that showed
the stronger hydrogen bond between pyrrole-NH and
protein and the lowest conformational energy
(ꢀ4860 kcal/mol) with respect to the mean value
60 kcal/mol less of all conformers collected during MD
simulation. The pyrrole-NH group of compound 11
interacts, by a strong hydrogen bond, with two different
amino acids of the receptor protein; depending on the
amino acid involved, molecule 11 can adopt two alterna-
˚
between acceptor and donor atoms is below 3.5 A.
For compound 8a (Fig. 3) those conformers of ligand–
protein complex that simultaneously exhibited strong
hydrogen-bond interactions involving the ethereal oxy-

4478
G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
Figure 2. Modes of binding (1–4) of each conformer (below example for conformer A).
tive modes of anchoring in the receptor pocket, arbi-
trarily called ‘binding mode type 1’ and ‘binding mode
type 2’ (Fig. 4a and b).
aTyr159, and the oxygen of the N-oxide group and the
N4-atom of benzotriazine system alternatively interact,
by strong hydrogen bond, with aSer205. The carbonyl
oxygen atom binds alternatively with cArg132 (NH gua-
nidine) or aTyr159 (hydroxyl group).
In ‘binding mode type 1’ (Fig. 4a), the pyrrole-NH inter-
acts with the amidic oxygen (side chain) of aGln203, and
the oxygen of N-oxide group alternatively interacts, by
strong hydrogen bond, with the hydroxyl group of
aTyr209 and of aSer205; the N4-atom of the benzotri-
azine system interacts with aTyr209 (hydroxy group).
The carbonyl oxygen atom binds alternatively with
cArg144 (NH guanidine) or aTyr159 (hydroxyl group).
The binding modes of compounds 8a and 11 permit us
to hypothesize why compound 11a (8-ethoxy-3-(1H-pyr-
rol-2-ylcarbonyl)pyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide) has a low affinity binding value (Ki = 1836 nM).
Since the driving force of interaction of compounds that
bear the 3-carbonylpyrrole moiety with receptor protein
is the strong hydrogen bond between the pyrrole-NH
and the corresponding amino acids (in both ‘binding
modes type 1 and 2’, vide supra), compound 11a would
In ‘binding mode type 2’ (Fig. 4b) the pyrrole-NH inter-
acts with the carbonyl oxygen (backbone CO) of

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4479
9b, 3-thenoyl derivative, was studied to evaluate how the
insertion of keto group influences the in vivo effect, with
respect to 3-thienyl derivative (lead, Chart 1) that
showed selective anticonvulsant activity.19 Six potential
benzodiazepine actions were considered: motor coordi-
nation was screened with the rota-rod test and the anti-
convulsant action was evaluated using the new drugs
against pentilenetetrazole-induced convulsions (Table
2); spontaneous motility and explorative activity with
hole-board test (Table 3); potential anxiolytic-like effects
were screened using light/dark choice test (Fig. 6);
mouse learning and memory modulation were evaluated
by passive-avoidance test (Fig. 7); and finally, the drugs
were tested also for their ethanol-potentiating action
(Fig. 8). Diazepam was used as the positive reference
molecule.
1.5. Effect on motor coordination
The effect of compounds 8a, 9b, and 11 was evaluated in
comparison with diazepam on mouse rota-rod test, in or-
der to point out motor incoordination activity (In Table
2 are reported only the pretest and the falls after 30 min
from treatment; The Complete data are reported in
Supplementary data (Table 4)). The reference compound
(diazepam 1 mg/kg ip) increased the number of falls from
the rotating rod, reaching statistical significance (Table
2). None of the newly synthesized substances induced
any effect on the number of mouse falls from the rota-
rod evaluated after 15, 30, 45, and 60 min (see Supple-
mentary data); moreover it is evidenced that mice learned
to stay on the rota-rod since the number of falls was
lower in time-dependent manner.
Figure 3. Docking of compound 8a, 3-(fur-2-ylcarbonyl)-8-ethoxypy-
razolo[5,1-c][1,2,4] benzotriazine 5-oxide: conformers with the lowest
conformational energy in the receptor protein.
anchor in the same manner. Unexpectedly compound
11a cannot bind the receptor protein in ‘1 or 2 type
modes’ due to steric hindrance of the 8-ethoxy group.
In fact in ‘binding mode type 1’ the substituent at posi-
tion 8 presents a strong repulsive interaction with
cThr81, while in ‘binding mode type 2’ the amino acids
cArg144 and aHis101 cannot act as hydrogen bond do-
nors (as hypothesized for 8-ethoxy group of compound
8a) because, probably, the 8-substituted benzotriazine
aromatic moiety gives steric hindrance (Fig. 5a and b).
1.6. Effect against chemically induced convulsions
Anticonvulsant activity was studied in mice using pen-
tilentetrazole (PTZ) as chemical convulsant agent: the
latency time (s) to shocks and convulsions was evalu-
ated. Diazepam (1 mg/kg ip) completely protected
against PTZ-induced shocks and convulsions (Table
2), while 8a only reduced (3 mg/kg po) the convulsion
1.4. In vivo pharmacology
Compounds 8a and 11 inserted on molecular modeling
studies were chosen also for in vivo testing. Compound
Figure 4. Docking of compound 11, 3-(1H-pyrrol-2-ylcarbonyl)-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide. Two alternative modes of
anchoring in the receptor protein: ‘binding mode type 1’ in (a) and ‘binding mode type 2’ in (b).

4480
G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
Figure 5. In panel (a) and (b) are evidenced amino acids not involved in binding of compound 11.
Table 3. Effect of compounds 8a, 9b, and 11 on spontaneous motility
and 9b were able to diminish curiosity at the dose of
and explorative activity in comparison of diazepam evaluated in the
mouse hole-board test
3 mg/kg po and 30 mg/kg po, respectively. By contrast
compound 11 at the dose of 3 and 10 mg/kg po did
not show any alteration of both spontaneous and
explorative activities while at the dose of 30 mg/kg po
it did diminish the panel movements (Table 3).
Treatmenta
mg/kg po
Hole board
n
Hole
Plane
CMC 1%
Diazepam
0.1 ml
3 (ip)
31
11
33.53 2.3
13.4 6.3**
66.36 1.6
49.7 6.6**
1.8. Effect on anxiety
8a
9b
11
3
10
10
10
19.4 0.5**
30.7 1.3
36.9 5.0
68.0 3.0
65.1 3.5
59.0 7.9
10
30
Effects on mouse anxiety of newly synthesized mole-
cules in comparison with diazepam were studied using
a light/dark box apparatus. In our experiments, com-
pound 9b showed a good anxiolytic-like effect at doses
of 10 and 30 mg/kg po with an efficacy comparable to
that shown by diazepam (Fig. 6). Also compound 11,
reveled an anxiolytic activity at the dose of 10 mg/kg
but not at the dose of 3 and 30 mg/kg po. The anxio-
lytic-like effects of 9b and 11 were completely antago-
nized by flumazenil (at a dose of 100 mg/kg ip, dose
at which flumazenil was able to antagonize the anxio-
lytic effect of diazepam) suggesting the agonist profile
at GABAA/BzR complex of the newly synthesized li-
gands (Fig. 6).
3
15
15
15
23.1 2.9
32.4 5.6
21.2 3.6*
62.8 8.0
73.0 7.9
52.2 11.5
10
30
3
17
17
17
29.2 3.0
39.1 2.2
33.5 2.2
67.1 2.8
68.5 3.1
56.3 2.0**
10
30
a The test was performed 30 and 20 min after the administration of
compounds (po) and the diazepam (ip), respectively.
* P < 0.05.
** P < 0.01 versus control mice.
latency. Compound 11 was devoid of any effect on
PTZ-shock and convulsion latency and compound 9b
increases the latency of both parameters in a significant
manner.
1.9. Effects on learning and memory
In order to clarify the effect that the newly synthesized
compounds have on learning and memory processes,
mice performance on passive-avoidance test was inves-
tigated. In this assay, the parameters taken into consid-
eration are represented by retention and training
latencies expressed in sec. While the training latencies
did not differ among the various groups, some retention
latencies are significantly different from the others. Par-
ticularly, as can be observed in Figure 7, compounds 9b
(10 and 30 mg/kg po) and 11 (3 and 10 mg/kg po) im-
proved, in a statistically significant manner, the mouse
memory processes. In the same experimental test the
full-agonist diazepam (1 mg/kg ip) showed an amnesic
activity.
1.7. Effects on spontaneous motility and curiosity
A very common effect of the old benzodiazepine ligands
is represented by sedation. The hole-board test is sensi-
tive in checking this kind of behavior as demonstrated
by the significant reduction of spontaneous motility
and curiosity exerted by diazepam at the dose of 3 mg/
kg ip.
Experiments carried out for compounds 8a, 9b, and 11
were finalized to evaluate their effects on mice spontane-
ous motility and explorative activity. Compounds 8a

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4481
180
160
140
120
100
80
***
***
***
***
^^^
^^^
60
40
20
0
mg/kg p.o.
CMC1%
1
100
3
10
3
10
30
10/100
3
10
30
10/100
Daz (ip) Flu (ip)
9b/Flu
8a
11
9b
11/Flu
Figure 6. Effect of compounds 8a, 9b and 11 in comparison with diazepam (Daz) on mouse light-dark box test. Columns represent the time, expressed
in s, spent in illuminated compartment. Treatment with compounds (po) and flumazenil (Flu) was performed 30 min and with diazepam 20 min
before the test. Each column represents means SEM of 14–21 mice. **P < 0.01, ***P < 0.001 versus controls. ^^^P < 0.001 versus 9b-treated or 11-
treated mice.
200
***
180
160
140
**
***
*
120
100
80
60
40
20
0
*
*
mg/kg p.o.
CMC1%
1
100
3
10
3
10
30
3
10
30
Daz (ip) Flu (ip)
11
8a
9b
Figure 7. Effect of compounds 8a, 9b, and 11 in comparison with diazepam (Daz) and flumazenil (Flu) on mouse passive-avoidance test. White
histograms represent the training latency and grey histograms represent the retention latency. Mice were treated 30 min before the training test. The
retention test was performed 24 h later. Each column represents means SEM of 15–18 mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus controls.
1.10. Effect on ethanol-induced sleeping time
Compounds 9b and 11 up to a dose of 300 mg/kg po
did not exhibit any modification of behavioral parame-
ters. Mice treated with 9b and 11 at the dose of
300 mg/kg po appeared on observation to be wholly
comparable to that of the control, to the extent that
researchers, who were unaware of the treatment received
by the animals, were unable to distinguish between the
various groups.
In the test of ethanol-induced sleeping time compounds
9b and 11 at the dose of 3, 10 and 30 mg/kg po increased
the duration of loss of righting reflex (Fig. 8). On the
contrary 8a did not modify this behavioral parameter.
As expected, the reference drug diazepam (daz, ip) en-
hanced sedation in a statistically significant manner.

4482
G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
160
140
120
100
80
***
***
***
***
***
***
***
60
40
20
0
mg/kg p.o.
CMC 1%
daz 1
3
10
3
10
30
3
10
30
Daz (ip)
8a
11
9b
Figure 8. Effect of compounds 8a, 9b, and 11 in comparison with diazepam (Daz) on mouse ethanol-induced sleeping time. Each column represents
means SEM of 12–14 mice. Substances were administered 30 min after ethanol (4 g/kg ip). ***P < 0.001 versus controls.
2. Conclusions
and/or N4 atom by means of a hydrogen bond involving
H-donor sites on receptor protein. Substituents at posi-
tions 3 and 8 interact with lipophilic pockets of receptor
proteins (L1/L2) and their influence on binding seem to
depend on electronic or chemical features. Moreover,
the interdependence between positions 3 and 8 was
shown, since the type of heteroaryl ring at position 3 en-
ables molecule to exert a further hydrogen bond with
receptor protein by ethereal oxygen at position 8. In fact
the presence of a ring able to be ‘hydrogen-bond accep-
tor’ toward receptor protein (furane oxygen in com-
pound 8a) ‘permits’ the molecule to engage another
hydrogen bond, through the ethereal oxygen of the 8-
substituent. On the contrary, the presence at position 3
of heteroaroyl ring able to be ‘hydrogen-bond donor’
(as 1H-pyrrol-2-yl, compound 11) shifts completely the
molecule in the receptor protein and two alternative
modes of anchorage can be suggested (Fig. 4a and b).
The pyrrole NH plays a crucial role in the receptor bind-
ing pocket because, first of all, it is involved in a strong
hydrogen bond in both anchorage modes and its alkyl-
ation determines a loss of affinity; second the coopera-
tive binding of pyrrole NH and N-oxide group is
evidenced since the N-deoxide ligand (11R) has low
affinity; this is in contrast with compound 9bR (3-the-
noyl derivative) which maintains the affinity value of
the corresponding 5-oxide derivative, 9b. On the other
hand an 8-alkyloxy substituent on 3-pyrrol-2-ylcarbonyl
derivative, 11a, is not tolerated probably because this
substituent can lie near to sterically forbidden sites
and the ethereal oxygen cannot form the hydrogen
bond, as instead happens for compounds 8a.
The synthesis of these compounds has permitted us to
further analyze the SAR for pyrazolo[5,1-c][1,2,4]benzo-
triazine ligands. Interesting confirmations that arise
from biological results concern the lipophilic pocket in
which the 3-substituent fits: the limited size and particu-
lar requisites are shown even in the presence of additive
carbonyl group with respect 3-heteroaryl derivative.19 In
fact, 3-aroyl derivatives with six-membered rings or
bulkier substituents (compounds 3, 4, 5, 5a, 6, 6a, 7)
show
a very low-affinity value (Ki range 919–
4200 nM). On the other hand the small methyl group
does not determine important increases on binding-
affinity (compound 2). The presence, instead, of elec-
tron-rich five-membered heteroaryl rings (as furoyl-,
thenoyl-, pyrroyl-), retains significant affinity to BzR.
In general the presence at position 8 of electron donat-
ing group as –OEt, –OPh, –OCH2Ph, and in position
3 of a furoyl- or thenoyl ring (compounds 8a–c and
9a–c) increase the affinity binding. By molecular model-
ing studies the increased binding of 8-alkoxy- and 8-
aryloxyderivatives would be due to an additional hydro-
gen bond that the ethereal oxygen could engage with
two amino acids (cArg144 or aHis101) on the receptor
protein.
Molecular modeling study strengthens our hypothesis of
ligand–receptor protein interaction,19 assuming as topo-
logical model the pharmacophore/model proposed by
Cook and co-workers51–54: the pyrazolobenzotriazine
system binds the GABAA/BZ complex through N1

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4483
From a pharmacological point of view, we might con-
sider 9b and 11 selective anxiolytic-efficacy ligands, with
a similar efficacy (for compound 9b) to that of diazepam
even if at a greater doses (10 mg/kg), but without its
undesirable side effect. The selective effect of 9b and 11
could be ascribed to an agonist-efficacy at a2-subtype;
moreover the activation of these subtypes could induce
an increase of loss righting reflex endurance,55 as evi-
denced for the same compounds (Fig. 8). On the other
hand ligands 9b and 11 not only show no impairment
on learning and memory mouse performance in statisti-
cally significant manner, but they are able to increase the
retention latency demonstrating their ability to amelio-
rate mnemonic processes. These data can lead to the
interpretation that compounds 9b and 11 could have
an antagonist profile at a1-containing receptor sub-
types-like flumazenil; in fact to these subtypes has been
attributed the amnesic effect of benzodiazepines.56 These
pharmacological aspects permit to distinguish the exam-
ined compounds from the old BDZ-ligands that were
endowed with amnesic properties.
opyrazol-4-yl)arylmethanones IV, V, IX and the en-
hydrazinyl derivatives III, VI, VII and VIII. Compound
III0, the 5-methylpyrazole-4-carbonitrile derivative, is
obtained by the treatment of the 2-nitro-5-chlorophenyl-
hydrazine30 and 2-ethoxymethylene-3-oxo butane nitrile
with ethanol/HCl.32
3.2.1. 2-Acetyl-3-(2-(5-chloro-2-nitrophenyl)hydrazinyl)
acrylonitrile (III). Orange crystals. TLC eluent: tolu-
ene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1
3369, 2203; 1H NMR (CDCl3) d 11.6 (d, 1H, NH exch.);
9.80 (s, 1H, NH); 8.20 (d, 1H, H-30); 7.57 (d, 1H, CH);
7.11 (d, 1H, H-60); 7.00 (dd, 1H, H-40); 2.50 (s, 3H,
CH3). Anal. C, H, N.
3.2.2. 1-(2-nitro-5-chlorophenyl)-5-methylpyrazole-4-car-
bonitrile (III0). Red-brown crystals. TLC eluent: toluene/
1
ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1 2232; H
NMR (CDCl3) d 8.13(d, 1H, H-30); 7.90 (s, 1H, H-3);
7.73 (dd, 1H, H-40); 7.54 (s, 1H, H-60); 2.45 (s, 3H,
CH3). Anal. C, H, N.
In summary, the introduction at position 3 of pyra-
zolobenzotriazine system of a heteroaroyl group, as in
Ocinaplon and Indiplon, lets us to individuate a new ser-
ies of GABAA/BZ receptor ligands, endowed with high
affinity. With respect to lead (anticonvulsant
activity, see Chart 1), the tested compounds show selec-
tive anxiolytic activity devoid of side effects. It is further
confirmed that the positions 3 and 8 of pyrazolobenzo-
triazine system are ‘key point’ to modify to obtain selec-
tive-efficacy ligands.
3.2.3. [5-Amino-1-(5-chloro-2-nitrophenyl)-1H-pyrazol-4-
yl](2-methoxyphenyl)methanone (IV). Dark-yellow crys-
tals. TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1
1
v/v/v; IR m cmꢀ1 3401, 3324, 1629; H NMR (CDCl3)
d 8.04 (d, 1H, H-30); 7.68 (d, 1H, H-60); 7.63 (dd, 1H,
H-40); 7.52 (s, 1H, H-3); 7.45 (m, 2H, H-400 and H-600
2-MeOPh); 7.05 (m, 2H, H-300 and H-500 2-MeOPh);
6.00 (br s, 2H, NH2 exch.); 3.90 (s, 3H, OCH3). Anal.
C, H, N.
3.2.4. [5-Amino-1-(5-chloro-2-nitrophenyl)-1H-pyrazol-4-
yl](pyridin-3-yl)methanone (V). Dark-yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
3. Experimental
3.1. Chemistry
1
v; IR m cmꢀ1 3401, 3324, 1624; H NMR (CDCl3) d
8.70 (dd, 1H, H-4 Py); 8.50 (d, 1H, H-2 Py); 8.10 (s,
1H, H-3); 8.08 (d, 1H, H-6 Py); 8.00 (d, 1H, H-30);
7.65 (dd, 1H, H-40); 7.45 (d, 1H, H-60); 6.20 (br s, 2H,
NH2 exch.). Anal. C, H, N.
Melting points were determined with a Gallenkamp
apparatus and were uncorrected. Silica gel plates (Merk
F254) and silica gel 60 (Merk 70–230 mesh) were used for
analytical and column chromatography, respectively.
The structures of all compounds were supported by their
IR spectra (KBr pellets in nujol mulls, Perkin-Elmer
3.2.5. 2-(1,3-Benzodioxol-5-yl)-3-(2-(5-chloro-2-nitro-
phenyl)hydrazinyl)acrylonitrile (VI). Yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
1
1
1420 spectrophotometer) and H NMR data (measured
with a Bruker 400 MHz). Chemical shifts were expressed
v; IR m cmꢀ1 3369, 2203; H NMR (CDCl3) d 12.00
(d, 1H, NH exch.); 10.00 (s, 1H, NH exch.); 8.20 (d,
1H, H-30); 7.80 (d, 1H, CH exch.); 7.70 (dd, 1H, H-
600); 7.48 (d, 1H, H-200); 7.20 (d, 1H, H-50); 7.00 (dd,
1H, H-40); 6.90 (d, 1H, H-500); 6.01 (s, 2H, CH2O). Anal.
C, H, N.
in d ppm, using DMSO-d6 or CDCl3 as solvent. The
coupling constant values (JH6–H7, H7–H6; JH7–H9,
H9–
H7;) were in agreement with the assigned structure.
The chemical and physical data of new compounds are
reported in Supplementary data. Microanalyses were
performed with a Perkin-Elmer 260 analyzer for C, H,
N, and the results were within 0.4% of the theoretical
value.
3.2.6. 2-(2-Furylcarbonyl)-3-(2-(5-chloro-2-nitrophenyl)
hydrazinyl)acrylonitrile (VII). Dark-yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
1
v; IR m cmꢀ1 3324, 2215, 1610; H NMR (CDCl3) d
3.2. General procedure for the synthesis of III–IX
11.90 (d, 1H, NH exch.); 9.85 (s, 1H, NH exch.); 8.22
(d, 1H, H-30); 8.05 (d, 1H, CH exch.); 7.70 (m, 2H, H-
5 and H-3 furane); 7.18 (d, 1H, H-60); 7.00 (dd, 1H,
H-40); 6.62 (dd, 1H, H-4 furane). Anal. C, H, N.
A
suspension of 2-nitro-5-chlorophenylhydrazine30
(2.0 mmol) and 2-ethoxymethylene-3-oxobutanenitrile32
or [(2-dimethylamino)methylene]-3-oxo-3-arylpropane-
nitrile33–36 (2.0 mmol) in ethanol (50 mL) and acetic acid
was refluxed until the starting material disappeared
(6 h). The work up of final solution gave the (5-amin-
3.2.7. 2-(2-Thienylcarbonyl)-3-(2-(5-chloro-2-nitrophenyl)
hydrazinyl)acrylonitrile (VIII). Dark-yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/

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1
v; IR m cmꢀ1 3345, 2213, 1609; H NMR (CDCl3) d
11.90 (d, 1H, NH exch.); 9.85 (s, 1H, NH exch.); 8.22
(d, 1H, H-30); 8.05 (d, 1H, CH exch.); 7.70 (m, 2H, H-
5 and H-3 thien.); 7.18 (d, 1H, H-60); 7.00 (dd, 1H, H-
40); 6.62 (dd, 1H, H-4 thien.). Anal. C, H, N.
3.3.3. 3-(Pyridin-3-ylcarbonyl)-8-chloropyrazolo[5,1-c][1,2,
4]benzotriazine 5-oxide (6). From V. Yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
1
v; IR m cmꢀ1 1626; H NMR (CDCl3) d 9.12 (d, 1H,
H-20 Py); 8.88 (dd, 1H, H-40 Py); 8.60 (s, 1H, H-2);
8.50 (m, 2H, H-6 and H-9); 8.25 (dd, 1H, H-60 Py);
7.70 (dd, 1H, H-7); 7.55 (dd, 1H, H-50 Py). Anal. C,
H, N.
3.2.8. [5-Amino-1-(5-chloro-2-nitrophenyl)-1H-pyrazol-4-
yl](thien-3-yl)methanone (IX). Ivory crystals. TLC elu-
ent: toluene/ethyl acetate 8:3 v/v; IR m cmꢀ1 3345,
1
2213, 1609; H NMR (CDCl3) d 8.06 (d, 1H, H-30);
3.3.4. 3-(1,3-Benzodioxol-5-ylcarbonyl)-8-chloropyrazol-
o[5,1-c][1,2,4]benzotriazine 5-oxide (7). From VI. Brown
crystals. TLC eluent: toluene/ethyl acetate/acetic acid
8.02 (d, 1H, H-60); 7.97 (s, 1H, H-2); 7.67 (m, 2H, H-
40 and H-20 thien.); 7.58 (m, 1H, H-30 thien.); 7.41 (m,
1H, H-400 thien.). Anal. C, H, N.
1
8:2:1 v/v/v; IR m cmꢀ1 1660; H NMR (CDCl3) d 8.50
(m, 3H, H-6, H-2 and H-9); 7.68 (dd, 1H, H-7); 7.50
(dd, 1H, H-60); 7.48 (d, 1H, H-20); 6.90 (d, 1H, H-50);
6.10 (s, 2H, OCH2O). Anal. C, H, N.
3.2.9. 3-Formyl-8-chloropyrazolo[5,1-c][1,2,4]benzotri-
azine5-oxide (1). To 6 mL of DMF maintained at 0ꢁC
was added POCl3 ( 0.8 mL, 8.6 mmol) and the solution
was stirred for 30 min; the starting material, 8-chloropy-
razolo[5,1-c][1,2,4]benzotriazine 5-oxide30 (4.0 mmol)
was slowly added. The reaction was maintained at
60 ꢁC for 48 h and monitored by TLC. Ice was added
into the brown solution and the formed precipitate
was filtered. The reaction mixture was purified by chro-
matography column using diisopropylether/cyclohexane
(8:3) as eluent and the eluted bands were characterized:
the 5-deoxide derivative 3-formyl-8-chloropyrazolo[5,1-
c][1,2,4]benzotriazine (faster eluted band), 1R, and
3-formyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide 1.
3.3.5. 3-(Fur-2-ylcarbonyl)-8-chloropyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (8). From VII. Yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
1
v; IR m cmꢀ1 1650; H NMR (CDCl3) d 8.85 (s, 1H,
H-2); 8.55 (d, 1H, H-6); 8.50 (d, 1H, H-9); 7.75 (d,
1H, H-50 fur); 7.68 (dd, 1H, H-7); 7.60 (d, 1H, H-30
fur); 6.68 (dd, 1H, H-40 fur). Anal. C, H, N.
3.3.6. 3-(Thien-2-ylcarbonyl)-8-chloropyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (9). From VIII. Brown crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
1
v; IR m cmꢀ1 1630; H NMR (CDCl3) d 8.62 (s, 1H,
H-2); 8.52 (d, 1H, H-6); 8.49 (d, 1H, H-9); 8.04 (d,
1H, H-30 thien.); 7.77 (d, 1H, H-50 thien.); 7.69 (dd,
1H, H-7); 7.23 (t, 1H, H-40 thien.). Anal. C, H, N.
Compound 1. Yellow crystals. TLC eluent: i-propyle-
ther/cyclohexane 8:3 v/v; IR m cmꢀ1 1677, 1565; 1H
NMR (CDCl3) d 10.28 (s, 1H, CHO.); 8.50 (m, 3H,
H-2, H-6 and H-9); 7.70 (dd, 1H, H-7). Anal. C, H, N.
3.3.7. 3-(Thien-3-ylcarbonyl)-8-chloropyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (10). From IX. Yellow crystals.
TLC eluent: petroleum benzine/ethyl acetate 31 v/v; IR
m cmꢀ1 1620, 1552; (CDCl3) d 8.62 (s, 1H, H-2); 8.53
(d, 1H, H-6); 8.50 (d, 1H, H-9); 8.29 (m, 1H, H-20
thien.); 7.73 (dd, 1H, H-50 thien.); 7.68 (dd, 1H, H-7);
7.42 (t, 1H, H-40 thien.). Anal. C, H, N.
Compound 1R. Yellow crystals. TLC eluent: i-propyle-
ther/cyclohexane 8:3 v/v; IR m cmꢀ1 1677; 1H NMR
(CDCl3) d 10.80 (s, 1H, CHO); 8.72 (m, 2H, H-2 and
H-6); 8.58 (d, 1H, H-9); 7.85 (dd, 1H, H-7). Anal. C, H, N.
3.3. General procedure for the synthesis of 2, 4, 6–10
3.4. General procedure for the synthesis of 3, 5, 11–13
The suitable intermediates III–IX (0.5 mmol) were sus-
pended in sodium hydroxide solution 10% and stirred
at 50 ꢁC for 36 hours. When the staring material disap-
peared the reaction was stopped and treated with water/
ice: the precipitate was filtered and recrystallized by a
suitable solvent.
The starting material, 3-carboxy-8-chloropyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide30 (0.4 mmol) was reacted
with 2.0 mL of thionyl chloride and maintained at 60 ꢁ
for 300; the final solution was evaporated in vacuo and
the corresponding 3-carbonylchloride intermediate was
utilized as raw material in Suzuki coupling for com-
pound 3 or in Friedel–Crafts reaction to obtain com-
pounds 5, 11–13. Compounds 8 and 9 were also
alternatively obtained by this synthetic procedure using
furane or tiophene as aryl compounds (yields shown on
Supplementary data).
3.3.1. 3-Acetyl-8-chloropyrazolo[5,1-c][1,2,4]benzotri-
azine 5-oxide (2). From III. Yellow crystals. TLC eluent:
toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1
1
1660; H NMR (CDCl3) d 8.60 (s, 1H, H-2); 8.52 (d,
1H, H-6); 8.47 (d, 1H, H-9); 7.67 (dd, 1H, H-7); 2.8 (s,
3H, CH3). Anal. C, H, N.
3.4.1. 3-Benzoyl-8-chloropyrazolo[5,1-c][1,2,4]benzotri-
azine 5-oxide (3). The raw intermediate 3-carbonyl chlo-
ride (0.4 mmol) was suspended in 8 mL of absolute
toluene. Phenylboronic acid (0.2 mmol), cesium carbon-
ate (0.5 mmol) and an excess of triphenylphosphine pal-
ladium(0) (Tetrakis) (10 mg) were added; the mixture
was maintained at refluxing temperature under nitrogen
3.3.2. 3-(2-Methoxybenzoyl)-8-chloropyrazolo[5,1-c][1,2,
4]benzotriazine 5-oxide (4). From IV. Yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v;
1
IR m cmꢀ1 1630; H NMR (CDCl3) d 8.48 (d, 1H, H-6);
8.46 (d, 1H, H-9); 8.45 (s, 1H, H-2); 7.64 (dd, 1H, H-7);
7.53 (m, 2H, H-40 and H-60); 7.10 (t, 1H, H-50); 7.04 (d,
1H, H-30); 3.75 (s, 3H, OCH3). Anal. C, H, N.

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4485
for 2 days. The final suspension was diluted with ethyl
acetate and washed with water and a saturated solution
of sodium bicarbonate. The ethyl acetate solution was
then dried over sodium sulfate and evaporated under
pressure. The residue was recrystallized by a suitable
solvent.
pyrr.); 6.32 (m, 1H, H-40 pyrr.); 4.10 (s, 3H, CH3-
N). Anal. C, H, N.
Compound 12 was also obtained by the alkylation of
compound 11: to a solution of DMF (10 mL) and 11
(0.13 mmol) were added anhydrous potassium carbon-
ate (0.13 mmol) and methyl iodide (0.26 mmol). The
reaction was maintained at 35 ꢁC and monitored by
TLC and when starting material disappeared water/ice
was added. The precipitate, compound 12, was filtered
and recrystallized by ethanol. Yield 15%.
Yellow crystals. TLC eluent: toluene/ethyl acetate/acetic
1
acid 8:2:1 v/v/v; IR m cmꢀ1 1677; H NMR (CDCl3) d
8.68 (s, 1H, H-2); 8.50 (m, 2H, H-6 and H-9); 7.68
(dd, 1H, H-7); 7.42 (m, 2H, H-20 and H-60); 7.25 (m,
3H, H-30, H-40 and H-50). Anal. C, H, N.
Compound 13. Yellow crystals. TLC eluent: toluene/
ethyl acetate 8:2 v/v; IR m cmꢀ1 1613; 1H NMR (CDCl3)
d 8.60 (s, 1H, H-2); 8.52 (d, 1H, H-6); 8.48 (d, 1H, H-9);
7.64 (dd, 1H, H-7); 7.52 (m, 1H, H-20 pyrr.); 6.82 (m,
1H, H-50 pyrr.); 6.67 (m, 1H, H-40 pyrr.); 3.80 (s, 3H,
CH3–N). Anal. C, H, N.
3.5. General procedure for Friedel–Craft reaction to
obtain compounds 5, 11–13
The raw intermediate 3-carbonyl chloride (0.4 mmol)
was suspended in a solution of 15 mL of methylene chlo-
ride and 0.8 mmol of anhydrous tin tetrachloride; the
red solution was maintained at reflux, then 1.2 mmol
of suitable aryl compounds were added. The reaction
was monitored by TLC and when the starting material
disappeared, the reaction was quenched by treatment
with HCl 1:1, diluted with methylene chloride and the
organic layer was separated. The methylene solution
was washed with a saturated solution of sodium bicar-
bonate, dried over sodium sulfate and evaporated. The
residue was purified by recrystallization.
3.6. General procedure for the synthesis of compounds 2a,
5a–6a, 8a–9a, 11a–12a
Starting compounds 2, 5–6, 8–9, 11, and 12 (0.1 mmol)
were suspended in ethanol (10 mL) and 10% sodium
hydroxide solution (5 mL) and were maintained at room
temperature for one day. The final suspension was fil-
tered or extracted with ethyl acetate. The precipitate
was recrystallized from a suitable solvent or the solution
was evaporated in vacuum and the residue purified.
3.5.1. 3-(4-Methoxybenzoyl)-8-chloropyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (5). From 3-carbonyl chloride
derivative and anisole; dark-yellow crystals. TLC eluent:
toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1
3.6.1. 3-Acetyl-8-ethoxypyrazolo[5,1-c][1,2,4]benzotri-
azine 5-oxide (2a). Yellow crystals. TLC eluent: toluene/
1
ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1 1663; H
1
NMR (CDCl3) d 8.58 (s, 1H, H-2); 8.47 (d, 1H, H-6);
7.72 (d, 1H, H-9); 7.23 (dd, 1H, H-7); 4.30 (q, 2H,
CH2); 2.8 (s, 3H, COCH3); 1.50 (t, 3H, CH3). Anal. C,
H, N.
1630; H NMR (CDCl3) d 8.50 (m, 3H, H-2, H-6 and
H-9); 7.94 (d, 2H, H-20 and H-60); 7.65 (dd, 1H, H-7);
7.01 (d, 2H, H-30 and H-50); 3.9 (s, 3H, OCH3). Anal.
C, H, N.
3.6.2. 3-(4-Methoxybenzoyl)-8-ethoxypyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (5a). Yellow crystals. TLC eluent:
toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1
3.5.2. 3-(1H-pyrrol-2-ylcarbonyl)-8-chloropyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (11). From 3-carbonyl
chloride derivative and pyrrole; yellow crystals. TLC
eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR
m cmꢀ1 3263, 1613; 1H NMR (CDCl3) d 9.92 (br s,
1H, NH exch.); 8.86 (s, 1H, H-2); 8.52 (d, 1H, H-6);
8.48 (d, 1H, H-9); 7.65 (dd, 1H, H-7); 7.33 (m, 1H, H-
50 pyrr.); 7.18 (m, 1H, H-30 pyrr.); 6.40 (m, 1H, H-40
pyrr.). Anal. C, H, N.
1
1629 H NMR (CDCl3) d 8.52 (s, 1H, H-2); 8.47 (d,
1H, H-6); 7.96 (d, 2H, H-20 and H-60); 7.75 (d, 1H, H-
9); 7.22 (dd, 1H, H-7); 7.01 (d, 2H, H-30 and H-50);
4.35 (q, 2H, CH2); 3.9 (s, 3H, OCH3); 1.60 (t, 3H,
CH3). Anal. C, H, N.
3.6.3. 3-(Pyridin-3-ylcarbonyl)-8-ethoxypyrazolo[5,1-c][1,2,
4]benzotriazine 5-oxide (6a). Yellow crystals. TLC elu-
ent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m
3.5.3. 3-(1-Methyl-1H-pyrrol-2-ylcarbonyl)-8-chloropy-
razolo[5,1-c][1,2,4]benzotriazine 5-oxide (12)
1
cmꢀ1 1630; H NMR (CDCl3) d 9.15 (d, 1H, H-20 Py);
3.5.4. 3-(1-Methyl-1H-pyrrol-3-ylcarbonyl)-8-chloropy-
razolo[5,1-c][1,2,4]benzotriazine 5-oxide (13). From the
reaction of 3-carbonyl chloride derivative and N-methyl-
pyrrole the two isomers were detected: purification by
chromatography column gave as faster band eluted
compound 12 and as second band the isomer (1-
methyl-1H-pyrrol-3-yl) derivative 13.
8.85 (dd, 1H, H-40 Py); 8.58 (s, 1H, H-2); 8.48 (d, 1H,
H-6); 8.20 (dd, 1H, H-60 Py); 7.80 (d, 1H, H-9); 7.50
(dd, 1H, H-50 Py); 7.19 (dd, 1H, H-7); 4.30 (q, 2H,
CH2); 1.50 (t, 3H, CH3). Anal. C, H, N.
3.6.4. 3-(Fur-2-ylcarbonyl)-8-ethoxypyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (8a). Yellow crystals. TLC eluent:
toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1
Compound 12. Yellow crystals. TLC eluent: toluene/
ethyl acetate 8:2 v/v; IR m cmꢀ1 1613; 1H NMR
(CDCl3) d 8.50 (m, 2H, H-2, H-6); 8.48 (d, 1H, H-
9); 7.64 (dd, 1H, H-7); 6.96 (m, 2H, H-30 and H-50
1
1650 H NMR (CDCl3) d 8.81 (s, 1H, H-2); 8.48 (d,
1H, H-6); 7.78 (d, 1H, H-9); 7.72 (d, 1H, H-50 fur);
7.63 (d, 1H, H-30 fur); 7.22 (dd, 1H, H-7); 6.66 (dd,

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G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
1H, H-40 fur); 4.30 (q, 2H, CH2); 1.60 (t, 3H, CH3).
Anal. C, H, N.
3.7.3. 3-(Thien-2-ylcarbonyl)-8-phenoxypyrazolo[5,1-c][1,2,
4]benzotriazine 5-oxide (9b). From 9 and phenol. Yellow
crystals. TLC eluent: toluene/ethyl acetate/acetic acid
1
3.6.5. 3-(Thien-2-ylcarbonyl)-8-ethoxypyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (9a). Yellow crystals. TLC eluent:
toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; IR m cmꢀ1
8:2:1 v/v/v; IR m cmꢀ1 1630, 1358; H NMR (CDCl3) d
8.55 (s, 1H, H-2); 8.53 (d, 1H, H-6); 8.05 (m, 1H, H-30
thien.); 7.73 (m, 2H, H-9 and H-50 thien.); 7.53 (t, 2H,
H-30 and H-50); 7.40 (t, 1H, H-40); 7.32 (dd, 1H, H-7);
7.20 (m, 3H, H-20, H-60 and H-40 thien.). Anal. C, H, N.
1
1630; H NMR (CDCl3) d 8.64 (s, 1H, H-2); 8.49 (d,
1H, H-6); 8.12 (m, 1H, H-30 thien.); 7.76 (m, 2H, H-9
and H-50 thien.); 7.24 (m, 2H, H-7 and H-40 thien.);
4.35 (q, 2H, CH2). Anal. C, H, N.
3.7.4. 3-(1H-pyrrol-2-ylcarbonyl)-8-phenoxypyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (11b). From 11 and phenol.
Yellow crystals. TLC eluent: toluene/ethyl acetate/acetic
acid 8:2:1 v/v/v; IR m cmꢀ1 3263, 1613; 1H NMR
(CDCl3) d 10.10 (br s, 1H, NH exch.); 8.62 (s, 1H, H-
2); 8.52 (d, 1H, H-6); 7.73 (d, 1H, H-9); 7.52 (t, 2H,
H-30 and H-50); 7.32 (m, 2H, H-40 and H-50 pyrr.);
7.20 (m, 4H, H-20, H-60 and H-30 pyrr.); 6.41 (m, 1H,
H-40 pyrr.). Anal. C, H, N.
3.6.6. 3-(1H-pyrrol-2-ylcarbonyl)-8-ethoxypyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (11a). Yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/
1
v; IR m cmꢀ1 3263, 1613; H NMR (CDCl3) d 10.20
(br s, 1H, NH exch.); 8.71 (s, 1H, H-2); 8.50 (d, 1H,
H-6); 7.75 (d, 1H, H-9); 7.41 (m, 1H, H-50 pyrr.); 7.22
(m, 2H, H-7 and H-30 pyrr.); 6.41 (m, 1H, H-40 pyrr.);
4.30 (q, 2H, CH2); 1.50 (t, 3H, CH3). Anal. C, H, N.
3.7.5. 3-(1-Methyl-1H-pyrrol-2-ylcarbonyl)-8-pheno-
xypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (12b).
From 12 and phenol. Yellow crystals. TLC eluent: tolu-
ene/ethyl acetate 8:3 v/v; IR m cmꢀ1 1613; 1H NMR
(CDCl3) d 8.52 (d, 1H, H-6); 8.42 (s, 1H, H-2); 7.72
(d, 1H, H-9); 7.64 (dd, 1H, H-7); 7.52 (t, 2H, H-30 and
H-50); 7.38 (t, 1H, H-40); 7.30 (dd, 1H, H-7); 7.20 (d,
2H, H-20, H-60); 6.96 (dd, 1H, H-50 pyrr.); 6.92 (m,
1H, H-30 pyrr.); 6.21 (m 1H, H-30 pyrr.); 4.10 (s, 3H,
CH3-N). Anal. C, H, N.
3.6.7. 3-(1-Methyl-1H-pyrrol-2-ylcarbonyl)-8-ethoxypy-
razolo[5,1-c][1,2,4]benzotriazine 5-oxide (12a). Yellow
crystals. TLC eluent: petroleum benzine/ethyl acetate
1
2:1 v/v; IR m cmꢀ1 1613; H NMR (CDCl3) d 8.50 (s,
1H, H-2); 8.47 (d, 1H, H-6); 7.75 (d, 1H, H-9); 7.20
(dd, 1H, H-7); 7.03 (m, 1H, H-30 pyrr.); 6.95 (m, 1H,
H-50 pyrr.); 6.25 (m, 1H, H-40 pyrr.); 4.35 (q, 2H,
CH2); 4.1 (s, 3H, NCH3); 1.50 (t, 3H, CH3). Anal. C,
H, N.
3.7. General procedure for the synthesis of compounds 2b,
8b–9b, 11b–12b, 8c–9c
3.7.6. 3-(Fur-2-ylcarbonyl)-8-benzyloxypyrazolo[5,1-c][1,2,
4]benzotriazine 5-oxide (8c). From 8 and benzyl alcohol.
Yellow crystals. TLC eluent: toluene/ethyl acetate 8:3 v/
1
A mixture of compounds 2, 5–6, 8–9, 11 ,and 12
(0.2 mmol), 10 mL of dichloromethane, 5 mL of 40% so-
dium hydroxide solution, 0.1 mole of tetrabutylammo-
nium bromide, and a large excess (5 mL) of the
suitable reagent (phenol or benzyl alcohol) was vigor-
ously stirred at 30–50 ꢁC for 4–12 h. The organic layer
was then separated and the aqueous layer extracted
twice with 10 mL of dichloromethane. The combined or-
ganic extracts were evaporated and the residue was
recovered with isopropyl ether and recrystallized by a
suitable solvent.
v; IR m cmꢀ1 1635; H NMR (CDCl3) d 8.82 (s, 1H, H-
2); 8.50 (d, 1H, H-6); 7.91 (d, 1H, H-9); 7.72 (d, 1H, H-50
fur); 7.63 (d, 1H, H-30 fur); 7.48 (m, 5H, Ph); 7.30 (dd,
1H, H-7); 6.67 (dd, 1H, H-40 fur); 5.38 (s, 2H, OCH2).
Anal. C, H, N.
3.7.7. 3-(Thien-2-ylcarbonyl)-8-benzyloxypyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (9c). From 9 and benzyl
alcohol. Yellow crystals. TLC eluent: toluene/ethyl ace-
tate 8:3 v/v; IR m cmꢀ1 1635; 1H NMR (CDCl3) d 8.63 (s,
1H, H-2); 8.50 (d, 1H, H-6); 8.11 (m, 1H, H-30 thien.);
7.90 (d, 1H, H-9); 7.75 (m, 1H, H-50 thien.); 7.52 (m,
5H, Ph); 7.32 (dd, 1H, H-7); 7.24 (m, 1H, H-40 thien.);
5.40 (s, 2H, OCH2). Anal. C, H, N.
3.7.1. 3-Acetyl-8-phenoxypyrazolo[5,1-c][1,2,4]benzotri-
azine 5-oxide (2b). From 2 and phenol. Yellow crystals.
TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v;
1
IR m cmꢀ1 1663, 1358; H NMR (CDCl3) d 8.56 (s, 1H,
3.7.8. 3-(Fur-2-ylcarbonyl)-8-propargyloxypyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (8d). From 8 and benzyl
alcohol. Yellow crystals. TLC eluent: toluene/ethyl ace-
tate 8:3 v/v; IR m cmꢀ1 1635; 1H NMR (CDCl3) d 8.82 (s,
1H, H-2); 8.51 (d, 1H, H-6); 7.92 (d, 1H, H-9); 7.73 (d,
1H, H-50 fur); 7.61 (d, 1H, H-30 fur); 7.30 (dd, 1H, H-7);
6.67 (dd, 1H, H-40 fur); 5.00 (s, 2H, OCH2); 2.70 (s, 1H,
CH). Anal. C, H, N.
H-6); 8.51 (d, 1H, H-2); 7.71 (d, 1H, H-9); 7.53 (t, 2H,
H-30 and H-50); 7.38 (t, 1H, H-40); 7.33 (dd, 1H, H-7);
7.20 (d, 2H, H-20 and H-60); 2.70 (s, 3H, COCH3). Anal.
C, H, N.
3.7.2. 3-(Fur-2-ylcarbonyl)-8-phenoxypyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (8b). From 8 and phenol. Yellow
crystals. TLC eluent: toluene/ethyl acetate 8:3 v/v; IR m
1
cmꢀ1 1650, 1358; H NMR (CDCl3) d 8.75 (s, 1H, H-
3.8. General procedure for the synthesis of compounds
9bR and 11R
2); 8.53 (d, 1H, H-6); 7.75 (d, 1H, H-9); 7.71 (d, 1H,
H-50 fur); 7.60 (d, 1H, H-30 fur); 7.53 (t, 2H, H-30 and
H-50); 7.38 (t, 1H, H-40); 7.32 (dd, 1H, H-7); 7.10 (d,
2H, H-20 and H-60); 6.64 (dd, 1H, H-40 fur). Anal. C,
H, N.
Starting materials, compounds 9b and 11, were treated
with triethylphosphite (TEP) in toluene abs as previ-
ously reported.38

G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
4487
3.8.1. 3-(Thien-2-ylcarbonyl)-8-phenoxypyrazolo[5,1-c][1,2,
4]benzotriazine (9bR). From 9b. Yellow crystals. TLC
eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v; H
NMR (CDCl3) d 8.70 (m, 2H, H-2 and H-6); 8.30 (dd,
1H, H-30 thien.); 7.82 (d, 1H, H-9); 7.78 (dd, 1H, H-50
thien.); 7.55 (m, 3H, H-30 and H-50 and H-7); 7.38 (m,
1H, H-40); 7.23 (m, 3H, H-20, H-60 and H-40 thien.).
Anal. C, H, N.
albino mice (22–24 g) and male Wistar rats (180–200 g)
(Harlan Italy) were used. Twelve mice and three rats
were housed per cage and fed a standard laboratory diet,
with tap water ad libitum for 12 h/12 h light/dark cycles
(lights on at 7:00). The cages were brought into the
experimental room the day before the experiment, for
acclimatization purposes. All experiments were per-
formed between 10:00 and 15:00.
1
3.8.2. 3-(1H-pyrrol-2-ylcarbonyl)-8-chloropyrazolo[5,1-
c][1,2,4]benzotriazine (11R). From 11; Yellow crystals.
TLC eluent: diisopropylether/ciclohexane 8:3 v/v; IR m
cmꢀ1 3263; 1H NMR (CDCl3) d 11.2 (br s, 1H, NH exch.);
8.81 (s, 1H, H-2); 8.65 (d, 1H, H-6); 8.52 (d, 1H, H-9); 7.78
(dd, 1H, H-7); 7.41 (m, 1H, H-50 pyrr.); 7.20 (m, 1H, H-30
pyrr.); 6.41 (m, 1H, H-40 pyrr.). Anal. C, H, N.
3.10.1. Rota-rod test. The integrity of the animals’ motor
coordination was assessed using a rota-rod apparatus
(Ugo Basile, Varese, Italy) at a rotating speed of 16
rpm. The treatment was performed before the test.
The numbers of falls from the rod in 30 s were counted
15, 30, 45 and 60 min after drug administration.
3.10.2. Hole-board test. The hole-board test was used to
evaluate the effects of drugs on a mouse’s spontaneous
motility and explorative capacity. Mice were placed indi-
vidually on the board and left free to explore both panel
and holes for 5 min, 30 min after drug administration.
3.9. Binding studies
[3H]Ro15-1788 (specific activity 70.8 Ci/mmol) was ob-
tained from NEN Life Sciences products. All the other
chemicals, which were of reagent grade, were obtained
from commercial suppliers.
3.10.3. Light/dark box test. The apparatus (50 cm long,
20 cm wide, and 20 cm high) consisted of two equal ac-
rylic compartments, one dark and one light, illuminated
by a 60 W bulb lamp and separated by a divider with a
10 · 3 cm opening at floor level. Each mouse was tested
by placing it in the centee of the lighted area, facing
away from the dark one, and allowing it to explore the
novel environment for 5 min. The time spent in the illu-
minated side was measured. This test exploited the con-
flict between the animal’s tendency to explore a new
environment and its fear of bright light.
Bovine cerebral cortex membranes were prepared as pre-
viously described.57 The membrane preparations were
diluted with 50 mM tris–citrate buffer, pH 7.4, and used
in the binding assay. Protein concentration was assayed
using the method of Lowry et al. [3H]Ro 15-1788 bind-
ing studies were performed as previously reported. After
removal, the cells were harvested by centrifugation at
500g. The crude membranes were prepared after homog-
enization in 10 mM potassium phosphate, pH 7.4, and
differential centrifugation at 48,000g for 30 min at
4 ꢁC. The pellets were washed twice in this manner be-
fore final resuspension in 10 mM potassium phosphate,
pH 7.4, that contained 100 mM potassium chloride.
[3H]Ro 15-1788 binding assays to transfected cell mem-
branes were carried out as previously described. In brief,
the cell line membranes were incubated in a volume of
500 ll, which contained [3H]Ro 15-1788 at a concentra-
tion of 1–2 nM and test compound in the 10ꢀ9–10ꢀ5 M
range. Non-specific binding was defined by 10ꢀ5 M diaz-
epam. Assays were incubated to equilibrium for 1 h at
4 ꢁC. The compounds were dissolved in DMSO, the le-
vel of which did not exceed 1% and which was main-
tained constant in all tubes. At least six different
concentrations of each compound were used. The data
of n = 5 experiments carried out in triplicate were ana-
lyzed by means of an iterative curve-fitting procedure
(program Prism, GraphPad, San Diego, CA), which
provided IC50, Ki, and SEM values for tested com-
pounds, the Ki values being calculated from the Cheng
and Prusoff equation.
3.10.4. Pentylenetetrazole (PTZ)-induced seizure. PTZ
(90 mg/kg sc) was injected 30 min after the administra-
tion of drugs. The number of clonic generalized convul-
sions was noted over a period of 30 min.
3.10.5. Passive-avoidance test. The test was performed
according to the step-through method described by Jar-
vik and Kopp.58 The apparatus consisted of a two-com-
partment acrylic box with a lighted compartment
connected to a darkened one by a guillotine door. As
soon as the mouse entered the dark compartment, it re-
ceived a thermal shock punishment. The latency times
for entering the dark compartment were measured in
the training test and after 24 h in the retention test.
The maximum entry latency allowed in the training
and retention sessions was, respectively, 60 and 180 s.
3.10.6. Ethanol-induced sleeping time test. Ethanol (4 g/
kg ip) was injected 30 min after drug administration.
The duration of a loss of the righting reflex was mea-
sured as the sleep time. The end-point was recorded as
150 min.
3.10. Pharmacological methods
The experiments were carried out in accordance with the
Animal Protection Law of the Republic of Italy, DL
No. 116/1992, based on the European Communities
Council Directive of 24 November 1986 (86/609/EEC).
All efforts were made to minimize animal suffering and
to reduce the number of animals involved. Male CD-1
3.10.7. Drugs. Diazepam (Valium 10–Roche), Flu-
mazenil (Tocris Cooksonl Ltd, UK), Pentylenetetrazole
(PTZ) (Sigma) were the drugs used. All drugs were dis-
solved in isotonic (NaCl 0.9%) saline solution and in-
jected sc/ip. All the new compounds were administered
by the po route and were suspended in 1% carboxymeth-

4488
G. Guerrini et al. / Bioorg. Med. Chem. 16 (2008) 4471–4489
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3.11. Molecular modeling
The model41 of the GABAA receptor was downloaded
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the BUILDER module of the INSIGHT II program.59
The complex ligand–protein was generated according
to the position of essential amino acids for ligands bind-
ing according to literature data.44–50
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To assess the preferred conformation of the ligand and in
the context of the selected conformer, a 100 ps molecular
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calculations were performed in vacuum with the DIS-
COVER module of the INSIGHT II program59 using
AMBER force field. A multiple-step procedure was used.
The complex was energetically minimized. The minimized
system was used as initial structure for the subsequent
molecular dynamics (MD) simulation. The system was
heated gradually until 310 K. Coordinates were saved
every 500 fs yielding 200 structures. The total time of com-
plex molecular dynamics simulation was 1.6 ns; during
the total dynamics simulation were collected 3200 confor-
mations. The dynamics simulation was carried out keep-
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Supplementary data
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Chemical data of intermediates III–IX (Table 1), of final
compounds (Table 2), analytical data (Table 3) and mo-
tor coordination table (Table 4). Supplementary data
associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2008.02.058.
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