Synthesis of novel cognition enhancers with pyrazolo[5,1-c][1,2,4] benzotriazine core acting at γ-aminobutyric acid type A (GABAA) receptor

Article abstract of DOI:10.1016/j.bmc.2013.02.027

Memory dysfunction associated with aging, neurodegenerative and psychiatric disorders represents an increasing medical need. Advances in research exploring the biological mechanisms underlying learning and memory have opened new potential approaches for development of memory-enhancing therapies addressed to selective neuronal targets. In this work, we synthesized some derivatives with a pyrazolo[5,1-c][1,2,4]benzotriazine core to identify ligands on GABA _A receptors subtype (benzodiazepine site on GABA_A- receptor) endowed with the potential of enhancing cognition activity without the side effects usually associated with non-selective GABA_A modulators. In fact, there is much evidence that GABA_A-R (γ-aminobutyric acid, type A receptor) subtype ligands have relevance in learning and memory. In vitro and in vivo tests have been performed. Pharmacological data indicate that compounds 7, 13, 14 and 22 act as dual functional modulators of GABA _A-Rs (promnemonic and anxiolytic agents) while only compounds 3 and 10 stand out as selectively displaying good antiamnesic and procognitive activity (1 and 3 mg/kg, respectively).

Full text of DOI:10.1016/j.bmc.2013.02.027

Bioorganic & Medicinal Chemistry 21 (2013) 2186–2198
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Bioorganic & Medicinal Chemistry
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Synthesis of novel cognition enhancers with pyrazolo[5,1-c][1,2,4]
benzotriazine core acting at
receptor
c-aminobutyric acid type A (GABAA)
a
a
b
b
Gabriella Guerrini a, , Giovanna Ciciani , Annarella Costanzo , Simona Daniele , Claudia Martini ,
⇑
Carla Ghelardini c, Lorenzo Di Cesare Mannelli c, Samuele Ciattini d
a Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente attivi (HeteroBioLab) Università degli Studi di Firenze,
Via U. Schiff, 6, 50019 Polo Scientifico, Sesto Fiorentino, Firenze, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università degli Studi di Pisa, via Bonanno, 6, 56126 Pisa, Italy
c Dipartimento di Farmacologia Preclinica e Clinica Aiazzi-Mancini, Università degli Studi di Firenze, Viale Pieraccini, 6, 50139 Firenze, Italy
d Centro di Cristallografia, Università degli Studi di Firenze, Via della Lastruccia, 3, 50019 Polo Scientifico, Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Memory dysfunction associated with aging, neurodegenerative and psychiatric disorders represents an
increasing medical need. Advances in research exploring the biological mechanisms underlying learning
and memory have opened new potential approaches for development of memory-enhancing therapies
addressed to selective neuronal targets. In this work, we synthesized some derivatives with a pyrazol-
o[5,1-c][1,2,4]benzotriazine core to identify ligands on GABAA receptors subtype (benzodiazepine site
on GABAA-receptor) endowed with the potential of enhancing cognition activity without the side effects
usually associated with non-selective GABAA modulators. In fact, there is much evidence that GABAA-R
Received 17 September 2012
Revised 16 January 2013
Accepted 14 February 2013
Available online 26 February 2013
Keywords:
Pyrazolo[5,1-c][1,2,4]benzotriazine system
GABAA receptors
(c-aminobutyric acid, type A receptor) subtype ligands have relevance in learning and memory. In vitro
and in vivo tests have been performed. Pharmacological data indicate that compounds 7, 13, 14 and 22
act as dual functional modulators of GABAA-Rs (promnemonic and anxiolytic agents) while only com-
pounds 3 and 10 stand out as selectively displaying good antiamnesic and procognitive activity (1 and
3 mg/kg, respectively).
Binding affinity
Cognition enhancing activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
attention of the pharmaceutical industry on mild cognitive impair-
ments (MCI) which significantly affect the quality of life.
Cognitive dysfunction is one of the most debilitating symptoms
of various acute or chronic neurological disorders. It may be a pro-
dromal phase of Alzheimer’s disease (AD)1–3 as well as a cardinal
feature in psychiatric conditions.4–6 Patients with Parkinson’s dis-
ease7,8 and Down syndrome9 also show alterations in learning
and memory. Since it is widely demonstrated that various neuro-
transmitters are involved in the considered diseases, multiple drug
therapy could be required. The ever-increasing elderly population
and resulting age-related neurological disorders have focused the
In the last decade a large amount of evidence suggests that the
-aminobutyric acid type A receptor (GABAA-R) could be a useful
c
target for memory enhancement.10,11 It is known that
c-aminobu-
tyric acid (GABA) is the major inhibitory neurotransmitter in the
mammalian central nervous system (CNS), acting through ionotro-
pic GABAA-Rs12 and metabotropic (G-protein coupled) GABAB
receptors (GABAB-Rs).
GABAA-Rs, which are members of the ligand-gated ion channels
(LGICs) superfamily, are membrane-bound heteropentameric pro-
teins composed principally of a, b and c subunits. At this time 21
subunits of the GABAA-Rs have been cloned13 and the most com-
mon are two single alpha subunits, two single beta subunits and
Abbreviations:
a7nACh-R, a7nicotinic acetylcoline receptor; AD, Alzheimer’s
one gamma subunit (
a
2
)2(b)2c 14
form
2) modulated by benzodiazepines (Bzs)
.
The combination of the isoforms
subset of GABAA-Rs 1b2 2,
disease; BMS, borane methylsulfide; Bz site/GABAA-R, benzodiazepine site on
GABAA receptor; CMC, carboxymethylcellulose; CNS, central nervous system; DAZ,
diazepam, 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2(1H)-one; Flu, flu-
mazenil (Ro15-1788); GABA, c-aminobutyric acid; GABAA-R, c-aminobutyric acid
type A receptor; LGCIs, ligand-gated ion channels; MCI, mild cognitive impair-
a1–3,5, b2/3 and
2b3 2, 3b3 2,
c
a
(a
c
a
c
a
c
a
5b3
c
and by ligands (also named non-benzodiazepine ligands) belong-
ing to several chemical classes. The Bz binding site is located at
ments; PTZ, pentylenetetrazole; SAR, structure affinity relationships.
⇑
Corresponding author. Tel.: +39 0554573766; fax: +39 0554573671.
E-mail address: gabriella.guerrini@unifi.it (G. Guerrini).
the interface of an
a and a c subunit and its pharmacology is
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.027

G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
2187
mainly determined by the alpha isoform.15–18 Genetic and
pharmacological approaches have identified the function of
GABAA-R subtypes. Receptors containing a1b2c2 subunits mediate
In the present study, lead compound 1 was optimized mainly
through classical isosteric replacement at position 8 or through
the introduction of 5-membered heterocycles (as triazole, or oxadi-
azole) in position 3. The binding affinities of the new compounds
were investigated and for preliminary pharmacological studies,
six compounds among the most representative (bearing different
chemical groups) were evaluated in vivo.
sedative, anterograde amnesic and anticonvulsant actions of
diazepam.19–21 The anxiolytic-like effect and muscle relaxant
activities are mediated by the
3-containing GABAA-Rs may be involved in the inhibitory input
for the dopaminergic system22–24 and the
5b 2 receptors may
influence learning and memory processes.18–25 The important role
of the 5b
2 GABAA-Rs, mainly localized in the hippocampus26,27
which is involved in cognitive functions, has been widely studied.
a2bc2 and a3bc2 receptors; the
a
a
c
2. Chemistry
a
c
All new pyrazolo[5,1-c][1,2,4]benzotriazine derivatives 2–6, 8,
10–25 here described are listed in Table 1 (chemical data). The
new 3-iodo-8-heteroarylmethylamino derivatives 2–5 were pre-
pared from compound I 3-iodo-8-chloropyrazolo[5,1-c][1,2,4]ben-
zotriazine 5-oxide42 treated with an excess of suitable
heteroarylalkylamine. The 3-iodo-8-(pyridin-4-ylmethoxy)pyraz-
olo[5,1-c][1,2,4]benzotriazine 5-oxide,41 our lead compound in this
work (1), was used as starting material for the synthesis of new
compound 6, its N-oxide derivative on the pyridine ring useful
for SAR (Scheme 1).
Compound II (3-carboxy-8-chloropyrazolo[5,1-c][1,2,4]benzo-
triazine 5-oxide43) was used as starting material to obtain the
3-methyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine derivative 8
(Scheme 2) using borane dimethylsulfide complex as reducing
agent.
This borane complex is reported to reduce the carboxy group to
methyl group.44 Compound 8 was then oxidized to the correspond-
ing N5-oxide 9, already reported in our previous work, but synthe-
sized with a more complex method and with minor yields.45 The
nucleophilic substitution on compound 9 with 4-pyridylmethanol,
2-chloro-4-hydroxymethylpyridine, and (2-(hydroxymethyl)furan)
was realized by phase transfer catalysis (PTC),41 and the com-
pounds 10–12 were, respectively obtained (Scheme 2).
The GABAA
a5 receptors’ density and function remain relatively
intact28,29 in the learning impairment in Alzheimer’s disease while
changes in many neurotransmitter receptors and overall choliner-
gic neurons are depleted during the disease. Several studies10,30
suggest that an a5-subtype selective inverse agonist could be pro-
cognitive without inducing convulsions and/or anxiety and many
different chemical classes endowed with selective affinity or selec-
tive efficacy have been identified.31–33 Compounds containing the
triazolopyridazine,34 the triazolophthalazine35 and the pyrazolotri-
azine30 core showed either high affinity or high inverse efficacy at
the
a5 subtype. During evolution of the triazolophthalazine
ligands, several bicyclic36,37 and tricyclic compounds were synthe-
sized38 and among them, the ligands
a5IA, a5IA-II and MRK-016
emerged.10,39,40
Our research group has been extensively involved in the design
of derivatives containing pyrazolo[5,1-c][1,2,4]benzotriazine
a
system, with the aim of identifying ligands which bind to the Bz
site on the GABAA-R with high affinity and selective pharmacolog-
ical activity as anxiolytic-like agents or promnemonic agents. In
our recent study the 3-iodo-8-(pyridin-4-ylmethoxy)pyrazolo
[5,1-c][1,2,4]benzotriazine 5-oxide 1,41 emerges as the compound
that improves mouse memory processes selectively, safely, in a
statistically significant manner, and in strict chemical relationship
The compounds bearing the oxadiazole or the triazole ring at
position 3 of the pyrazolobenzotriazine system were prepared
to
a5IA,
a5IA-II and MRK-016 (Chart 1).
N
N
3
Ki value: 1.3 0.1nM
O
N
I
8
Promnemonic propertiesat 3 and 10mg/Kg
Passive avoidance test
N
N+
O-
1
N
N
N
N
N
N
N
CH3
CH3
CH3
N
N
O
N
N
N
O
N
O
N
N
H3C
H3C
O
N
O
O
H3C
N
N
N
N
H3C
N
CH3
N
α5IA
α5IA-II
Chart 1.
MRK-016

2188
G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
Table 1
Chemical data for new synthesized compounds
N
R
N
R
8
3
N
+
-
N
O
No.
R3
R8
Yield (%)
Mp (°C) (recryst. solvent)
1a
2
3
4
5
I
I
I
I
I
I
I
OCH2-4-Py
—
NHCH2-4-Py
NHCH2-3-Py
NHCH2-2-Py
NHCH2-2-furyl
OCH2-4-Py-N-oxide
OCH2-2-thienyl
25
20
19
75
60
220–222 (Ethanol)
230 dec (Ethanol)
205 dec (Ethanol)
210–211 (Ethanol)
236–237 (Methoxyethanol)
—
156–157 (Ethanol)
—
6
7a
8
Me
Me
Me
Me
Cl
Cl
60
23
50
25
50
79
89
87
78
80
87
80
88
72
95
80
80
70
9b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
OCH2-4-Py
241–242 (Methoxyethanol)
276 dec (Ethanol)
OCH2-2-Cl-pyridin-4-yl
OCH2-2-furyl
OCH2Ph
OCH2-4-Py
OCH2-2-thienyl
OCH2Ph
OCH2-4Py
OCH2-2-thienyl
OCH2Ph
OCH2-4-Py
OCH2-2-thienyl
OCH2Ph
OCH2-4-Py
OCH2Ph
Me
296–297 (Ethanol)
240–241 (Isopropyl alcohol)
223–224 (Methoxyethanol)
240 dec (Methoxyethanol)
239–240 (Methoxyethanol)
240–241 (Methoxyethanol)
229–230 (Methoxyethanol)
209–210 (Toluene)
250–251 (Toluene)
235–236 (Toluene)
259–260 (Methoxyethanol)
269–270 (Methoxyethanol)
235–236 (Methoxyethanol)
255 dec (Ethanol)
1,2,4-Oxadiazol-3-Me-5-yl
1,2,4-Oxadiazol-3-Me-5-yl
1,2,4-Oxadiazol-3-Me-5-yl
CONH2
CONH2
CONH2
CO–N@CH –N(Me)2
CO–N@CH –N(Me)2
CO–N@CH –N(Me)2
1,2,4-Triazol-3-yl
1,2,4-Triazol-3-yl
1-Me-1H-1,2,4-triazol-5-yl
1-Me-1H-1,2,4-triazol-3-yl
OCH2Ph
a
See Ref. 41.
See Ref. 45.
b
N
N
Cl
N
R
NH
N
I
8
I
3
i
N°
2
R
CH2-4-Py
CH2-3-Py
CH2-2-Py
CH2-2-furyl
N
3
N
N+
O-
N+
O-
4
5
Ia
2-5
N
O-
N+
N
O
N
I
N
O
N
I
N
N+
O-
N
ii
N+
O-
1b
6
Scheme 1. aSee Ref. 42; bsee Ref. 41. Reagents and conditions: (i) EtOH, 50 °C, heteroarylalkylamine; (ii) H2O2 30%/AcOH.
according to two synthetic pathways (Scheme 3A and B), depend-
ing on the yields or the best reaction work-up for the obtaining of
the desired compounds.
To synthesize the derivatives 13–15 (route A), the first step was
to transform the 3-amide group of compound III, 3-carboxamido-
8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide,43 into the
dimethylaminovinyl derivative IV.46 Then the next cyclization with
NH2OHꢀHCl gave the corresponding 3-(1,2,4-oxadiazol-3-methyl-
5-yl) derivative V.46 In turn, this compound represented the start-
ing material for the nucleophilic substitution of the 8-chlorine
atom by PTC. The suitable alcohol is added to a two-phase system
(strong aqueous sodium hydroxide solution (40%) and methylene
chloride with tetrabutylammonium bromide (NBu4+Brꢁ) as cata-
lyst) and compounds 13–15 were obtained.
To obtain the 3-(1,2,4-triazol)-3-yl derivatives, we used route B
(Scheme 3), in which the nucleophilic substitution of 8-chlorine on

G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
2189
N
N
Cl
N
Cl
N
COOH
CH3
i
N
N
N+
O-
N
8
IIa
ii
N
N
R
O
N
CH3
Cl
N
CH3
iii
N
N+
O-
N
N+
O-
10 -12
9
N°
R
10 CH2-4-Py
11 CH2- 2 -Cl-Pyrid-4-yl
12 CH2-2-Furyl
Scheme 2. aSee Ref. 43. Reagents: (i) BH3ꢀS(CH3)2 complex; (ii) H2O2 30%/AcOH; (iii) R-OH, NaOH 40% solution, NBu4+Brꢁ/CH2Cl2.
compound III43 was the first step of synthesis and compounds 16–
18 were obtained. Then the treatment of 16–18 with dimethyl-
formamide–dimethylacetal (DMF–DMA) gave the corresponding
acylamidines 19–21, key intermediates for the synthesis of 3-tria-
zole derivatives. Only intermediates 19 and 20 cyclized with
hydrazine hydrate at 90 °C, gave the corresponding 22 and 23,
while the desired derivative was not obtained from compound
21 even by modifying the reaction conditions.
(Ro15-1788) from its specific binding in bovine brain membrane
and was expressed as Ki value only for those compounds inhibiting
radioligand binding by more than 80% at fixed concentrations of
10
l
M.
The binding data (Table 2) show that most of the new synthe-
sized compounds have receptor recognition (except 24 and 25)
and that some compounds (2–5) were able to bind to Bz site/GA-
BAA-R with affinity value (Ki) in a range of 0.2–0.7 nM.
To better elaborate the structure affinity relationships (SAR), we
decided to methylate the 1,2,4-triazole ring of compound 22 with
MeI. Street38 and Del Giudice47 reported that with NaH as alkaline
medium the two regioisomers N1 and N2 were prevalently ob-
tained, while the N4 methylation was not very easy to realize in
these conditions.48,49
The isosteric substitution O/NH, at 8-position in the lead com-
pound 1, afforded compound 2 endowed with an excellent affinity
value (2 Ki = 0.331 vs 1, Ki = 1.3 nM), maintained also in isomeric
compounds 3 and 4, the 3- and 2-pyridil derivatives, (Ki = 0.332
and 0.717 nM, respectively). The substitution of the pyridine ring
in compound 4 with a furyl ring gives compound 5 with Ki va-
lue = 0.23 nM showing that the introduction of a 5-membered het-
erocycle is again positive. On the basis of binding data, we observe
that compounds 2–5 show the best affinity values, confirming pre-
viously reported data45 for the arylalkylamino derivatives. In fact,
at position 8, the NH moiety (hydrogen bond donor) versus O
(hydrogen bond acceptor) plays a key role in receptor recognition,
engaging a hydrogen bond interaction stronger than the oxygen
atom (data not reported).
In our synthetic procedure (NaH/DMF/MeI), as expected, a mixture
of two isomers in a ratio of about 3/1 (assigned by NCH3 peaks at 4.051
and 4.153 ppm in 1H NMR spectroscopy) was recovered and only one
was obtained as pure product (isomer with the NCH3 signal at
4.051 ppm) by crystallization. Unfortunately, an X-ray analysis was
not performed because unsuitable crystals were obtained and it was
impossible to assign the isomer structure using only 1H NMR spectros-
copy. Thus, to determine exactly which isomer was obtained, a differ-
ent synthetic approach and different reaction conditions were used. By
reaction of 19 with methylhydrazine (Scheme 4) we obtained only
the isomer 3-(1,2,4-triazol-1-methyl-5-yl)-8-benzyloxypyrazolo
[5,1-c][1,2,4]benzotriazine 5-oxide 24 whose structure was deter-
mined by crystallographic data (Fig. 1). Since the NCH3 peak of 24 falls
at 4.153 ppm, compound 24 is not the same isomer obtained by crys-
tallization in the previous reaction. Thus, obviously, we can assign to
this latter compound the structure of 3-(1,2,4-triazol-1-methyl-3-yl)-
8-benzyloxypyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 25. However,
to try to synthesize unambiguously the isomer 25 a literature proce-
dure48 was followed choosing lithium hydroxide as alkaline medium.
Thus 22 was alkylated with MeI in LiOH and only the regioisomer 25
was obtained.
Compound 6, the N-oxide derivative of the lead compound,
shows an affinity value reduced fourteen times (Ki = 20.4 nM) with
respect to 1, whereas when the pyridine ring was substituted with
a thienyl ring, as in compound 7, comparable affinity value was
maintained (Ki = 1.2 nM).
The substitution on lead compound 1 of the iodine atom with
a methyl group gave compound 10 with affinity binding 24-fold
lower (10 Ki = 24.1 nM vs 1 Ki = 1.3 nM), while its 2-chloroderiva-
tive 11 has a good affinity value (Ki = 7.4 nM). The substitution of
the pyridine ring with a furan moiety gave comparable affinity
value to 10 (Ki = 30.9 nM). Despite the reduced ability of the
methyl group to form a lipophilic interaction with a receptor,
(methyl,
p = 0.56 and iodine p
= 1.12)50 the introduction of the
heteroaromatic ring at position 8 gave a good hydro/lipophilic
balance.
3. Result and discussion
3.1. In vitro binding
The substitution of iodine, at position 3 in the lead compound,
with different 5-membered heterocycles, produced compounds
13–15 and 22–23 with Ki values in a range of 70.7–1623 nM. The
methylation of compound 22 gave the two regioisomers 24 and
25 that are lacking receptor recognition, evidencing the key role
The Bz site/GABAA-R binding affinity of newly synthesized com-
pounds was evaluated by their ability to displace [3H]flumazenil

2190
G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
N
Cl
N
RouteA
Route B
CONH
2
N
+
-
N
i
ii
O
IIIa
N
N
NMe
2
N
N
Cl
H
RCH O
N
2
CONH
O
2
N
+
N
N
+
-
N
-
O
N°
R
IVb
O
16-18
16 Ph
17 4-Py
18 2-thienyl
iii
ii
N
N
O
N
Cl
N
N
NMe
2
N
RCH O
2
N
N°
R
CH
N
+
3
N
H
19 Ph
N
O
20 4-Py
21 2-thienyl
+
-
N
O
Vb
-
O
19-21
i
iii
H
N
N
N
N
N
RCH O
2
N
O
N
RCH O
2
N
N
+
-
N
N
CH
N
+
3
N
O
22-23
-
O
13-15
N°
R
N°
R
22 Ph
13 Ph
23 4-Py
14 4-Py
15 2-thienyl
Scheme 3. aSee Ref. 43; bsee Ref. 46. Reagents and conditions: (i) R-OH, NaOH 40% solution, NBu4+Brꢁ/CH2Cl2; (ii) DMF/DMA for 19–21 and DMA–DMA for IV; (iii)
NH2NH2ꢀH2O, EtOH, 90 °C.
of triazole NH as hydrogen bond donor or the steric hindrance of
the methyl group.
We also tested the intermediates 16–21 useful for the synthesis
of these latter derivatives and they show affinity in the range of
323–1200 nM. Compound 13 results in being the best ligand with
a Ki value of 70.7 nM.
learning) and some potential benzodiazepine actions were consid-
ered (anticonvulsant activity, potential anxiolytic-like effects and
the effect on motor coordination).
3.3. Passive avoidance test
In order to investigate the effect of compounds 3, 7, 10, 13, 14 and 22
on learning and memory, mouse performance in the passive avoidance
test (an experiment in which the animal learns to avoid a noxious event
by suppressing a particular behavior) was evaluated.
Examining the results, we can say that the training session la-
tency values did not differ for the various compounds, while the
3.2. In vivo activity
Compounds 3, 7, 10, 13, 14 and 22 were chosen for in vivo tests
in animal models, using various rodent species (mice and rats). The
effects on learning and memory (passive avoidance test and social

G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
2191
H
N
N
N
BnO
N
N
N
+
-
N
O
22
iii
i
CH
N
3
N
N
N
CH
N
3
BnO
N
N
BnO
N
+
N
N
N
N
+
-
+
-
N
N
O
O
24
25
ii
CH
3
N
N
CH
N
3
BnO
N
H
O
N
+
-
N
O
19
Scheme 4. Reagents: (i) NaH/DMF, MeI; (ii) MeNHNH2/AcOH; (iii) LiOH/MeI/EtOH.
To evaluate the antiamnesic effect of the compounds, (their
capability to prevent the amnesic effect of scopolamine (1.5 mg/
kg)), we tested them at the dose at which they were completely de-
void of promnemonic activity (7, 10, 13 and 22 at 1 mg/kg; 14 at
3 mg/kg and 3 at 0.1 mg/kg) in passive avoidance test with scopol-
amine (Fig. 2). All compounds were able to completely prevent the
amnesic effect of scopolamine.
3.4. Social learning
In the social learning test adult rats were treated with com-
pounds
3 (0.3–10 mg/kg po), 7 (10–100 mg/kg po), 10 (1–
10 mg/kg po), 13, 14, and 22 (10 mg/kg po) and the duration
of exploration of the familiar partner in the second session
was shortened in comparison with carboxymethylcellulose
(CMC)-treated rats; positive control: piracetam (30 mg/kg po
(Table 3). No curtailment was observed if an unknown partner
was presented to adult rats (data not shown). No compound
modified the duration of active exploration in the first session
in comparison with CMC-treated rats. All compounds exhibited
comparable efficacy even if the highest potency was exhibited
by 3 (1 mg/kg) and the lowest by 7 (30 mg/kg). The shortening
of the exploring time of the familiar rat induced by 3, 7, 10,
13, 14 and 22 in the social learning test indicated procognitive
activity in the absence of amnesia.
Figure 1. X-ray structure of compound 24.
retention latencies differed for some products (depending on the
doses). As depicted in Figure 2, mouse performances were tested
with compounds 3 (0.1, 1, 3 and 10 mg/kg po), 7 (1, 10, 30 and
100 mg/kg po), 10 (1, 3 and 10 mg/kg po), 13 (1, 3, 10 and
30 mg/kg po), 14 (3, 10 and 30 mg/kg po), 22 (1, 3 and 10 mg/kg
po). All tested compounds produced no amnesic effect, but im-
proved mouse memory processes by increasing the retention la-
tency compared to control in a statistically significant manner.
The most interesting results were recorded for 3 at 1 mg/kg, for
10 and 22 at 3 mg/kg, and for 13 at 10 mg.

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G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
Table 2
kg po), 7 (30 mg/kg po), 10 (3 and 10 mg/kg po), 13, 14 and 22 (3,
BZR ligand affinity of new compounds
10 and 30 mg/kg po) (Fig. 3). Compounds 13, 14 and 22 at the
doses of 10 and 30 mg/kg po and 7 at the dose of 30 mg/kg po re-
vealed an anxiolytic effect, comparable to diazepam, in a statisti-
cally significant manner. The anxiolytic effect of compounds was
completely antagonized by flumazenil (at a dose of 100 mg/kg
po), a dose at which flumazenil was able to antagonize the anxio-
lytic effect of diazepam (data not shown). Compounds 3 and 10
showed no activity in this experimental model.
N
R
8
N
R
3
N
+
-
N
O
No. R3
R8
I%a or Kib (nM)
3.6. Rota-rod test
1c
2
I
I
OCH2-4-Py
1.30 0.10
0.33 0.08
0.33 0.07
0.72 0.06
0.23 0.02
20.4 2.9
1.20 0.10
400.0 11.0
315.0 20.0
24.1 2.0
NHCH2-4-Py
NHCH2-3-Py
NHCH2-2-Py
NHCH2-2-furyl
OCH2-4-Py-N-oxide
OCH2-2-thienyl
The effects of compounds 3 and 10 (10 mg/kg po), 7, 13, 14 and
22 (30 mg/kg po) were studied in the mouse rota-rod test, in com-
parison with diazepam (10 mg/kg po), in order to determine any
motor incoordination activity. Table 4 reports the pretest and the
falls 15, 30, 45, 60 min after the treatment. None of the considered
compounds induced any effect on the number of mouse falls from
the rota-rod, confirming that mice learned to stay on the rota-rod
since the number of falls decreased in a time-dependent manner.
Protection from convulsions was evaluated in mice using penty-
lentetrazole (PTZ) as chemical convulsant agent. All compounds
were tested at the doses of 10 mg/kg po, except 3 that was tested
at 1 mg/kg po. Diazepam (10 mg/kg po) completely protected
against PTZ-induced shocks and convulsions. These compounds
were devoid of any effect on PTZ-induced shocks and convulsions
(Table 5).
3
I
4
I
5
I
6
I
I
7c
8
Me
Cl
Cl
9d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Me
Me
Me
Me
OCH2-4-Py
OCH2-2-Cl-pyridin-4-yl 7.49 0.19
O-CH2-2-furyl
OCH2Ph
OCH2-4-Py
OCH2-2-thienyl
OCH2Ph
OCH2-4-Py
OCH2-2-thienyl
OCH2Ph
OCH2-4-Py
OCH2-2-thienyl
OCH2Ph
30.9 3.0
70.7 5.7
1,2,4-Oxadiazol-3Me-5-yl
1,2,4-Oxadiazol-3Me-5-yl
1,2,4-Oxadiazol-3Me-5-yl
CONH2
CONH2
CONH2
CO–N@CH –N(Me)2
CO–N@CH –N(Me)2
CO–N@CH –N(Me)2
1,2,4-Triazol-3-yl
1,2,4-Triazol-3-yl
453.2 53.7
1623.0 150.9
323.7 33.0
1200.0 118.0
959.6 77.8
86.6 13.2
514.8 78.2
620.0 54.2
325.2 30.0
167.9 15.1
46%
3.7. Interaction with GABAA receptor subtypes
OCH2-4-Py
1-Me-1H-1,2,4-triazol-5-yl OCH2Ph
1-Me-1H-1,2,4-triazol-3-yl OCH2Ph
Based on the above pharmacological results, we decided to ver-
ify the interaction of compounds 3 and 10, that show selective ant-
iamnesic and promnemonic activity, with GABAA receptor
50%
Percent of inhibition of specific [3H]Ro15-1788 binding at 10
lM concentration
are means SEM of five determinations.
a
subtypes (
5-subtype receptors with an affinity value in the nanomolar
range (Ki = 0.449 and 24.9 nM, respectively) and showed a percent
of inhibition (I% = 37) for the 2-subtype receptor. Surprisingly,
compound 10 was able to bind only the 1-subtype receptor
(Ki = 22.7 nM) and had no affinity for 2- and 5-subtype receptors
(Table 6). Binding to the GABAA-R subtype indicated that com-
pound 3 has subtype affinity for 5 that could explain its in vivo
efficacy in memory tasks. Instead, compound 10, despite its activ-
ity in the mouse/rat memory task, has no affinity for the 5-sub-
type and thus involvement of the GABA system is excluded.
a1, a2, a5). Compound 3 was able to bind the a1- and
b
Ki value are means SEM of five determinations.
See Ref. 41.
See Ref. 45.
c
a
d
a
a
a
3.5. Light–dark box
a
The effects of compounds 3, 7, 10, 13, 14 and 22 on mouse anx-
iety, compared with diazepam (10 mg/kg po), were studied using a
light/dark box apparatus. All compounds were tested starting from
the lowest dose at which they have procognitive activity: 3 (1 mg/
a
a
Figure 2. Effect of new compounds in Mouse Passive Avoidance test. All drugs were administered po 30 min before training session with the exception of scopolamine that
was injected immediately after punishment. Each value represents the mean of 10–12 mice. The first column represents the training session of CMC-treated group. ^P <0.05
and ⁄P <0.01, versus control mice. §P <0.01 versus scopolamine-treated mice.

G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
2193
Table 3
Effect of compounds 3, 7, 10, 13, 14, 22 in rat social learning test
Table 4
Effect of 3, 7, 10, 13, 14, 22 in the mouse rota-rod test
Compounds
Dose (mg/kg) po
First session
Second session
D
Treatment
Falls from rotating rod after treatment
Treatment
CMC 1%
3
Compounds Dose (mg/kg)
po
Pre-test
15 min
30 min
45 min
0.1 ml
0.3
1
10
10
30
100
1
63.1 6.1
58.3 8.4
65.3 3.1
63.4 7.5
68.3 8.5
65.9 7.3
67.7 6.6
62.7 7.3
63.4 5.2
65.1 7.3
69.8 6.6
66.3 7.7
65.8 7.1
68.2 7.3
35.3 3.3
28.9 6.1
21.6 4.2^
20.9 3.7^
35.8 3.2
23.6 4.1^
25.8 4.9^
29.5 5.5
20.2 4.3^
23.7 4.4^
26.7 3.8^
21.7 3.6^
25.5 4.3^
21.9 5.1^
27.6
29.4
43.7
42.5
32.5
42.3
41.9
33.2
43.2
41.4
43.1
44.6
40.3
46.3
CMC 1%
3
0.1 mL
10
6.1 0.5 5.5 0.5
6.3 0.5 4.9 0.4
5.8 0.4 5.1 0.3
5.9 0.5 5.2 0.4
5.9 0.5 5.4 0.5
6.3 0.6 5.3 0.5
6.0 0.4 5.3 0.4
3.2 0.4
3.1 0.3
3.0 0.2
3.0 0.4
3.4 0.3
3.6 0.4
3.4 0.4
2.5 0.4
2.4 0.4
2.8 0.3
2.8 0.4
2.7 0.4
2.4 0.5
2.9 0.3
7
30
7
10
10
13
30
14
30
10
22
30
3
Diazepam
10
6.3 0.3 6.4 0.3^ 6.4 0.5⁄ 5.6 0.4⁄
10
10
10
10
30
Each value represents the mean of five mice. ^P <0.05, ⁄P <0.01 in comparison with
CMC treated mice.
13
14
22
Piracetam
^
P <0.05 versus control rats. Tests was performed 30’ after drug administrations.
to bind the
in the nanomolar range (Ki = 0.449 and 24.9 nM, respectively)
and shows an inhibition percent (I% = 37) to 2-subtype receptor.
Compound 10, surprisingly, was able to bind only 1-subtype
receptors (Ki = 22.7 nM) and shows no affinity for 2- and 5-sub-
type receptor. From these biological results, compound 3 has sub-
type affinity for 5 that could explain its in vivo efficacy on
memory tasks. Instead, 10, despite its activity on mouse/rat mem-
ory tasks, has no affinity for 5-subtype and thus another neuro-
transmitter, rather than GABA, could be involved. A preliminary
study showed that 10 enhances the percentage of ACh release
and the maximum effect was reached 60–80 min after the admin-
istration. This aspect will be the object of our next research.
Another important result was obtained for compounds 13, 14
and 22, which possess both promnemonic- and anxiolytic-like ef-
fects in the range of 10–30 mg/kg. Elucidation of this dual profile
could be an attractive research starting point for neurological dys-
functions where both cognitive enhancement and anxiolysis are
required.
a1- and a5-subtype receptors with an affinity value
Each value represents the mean of six rats.
a
a
3.8. Effect on ACh release
a
a
As a preliminary study, the release of ACh was evaluated and, as
shown in Figure 4, a statistically significant increase of ACh release
in freely moving rats was induced by administration of compound
10 (10 mg/kg ip), which peaked 60–80 min after administration
and returned to basal values within 120 min.
a
a
4. Conclusion
A series of new 3,8 disubstituted pyrazolo[5,1-c][1,2,4]benzotri-
azine derivatives were designed and synthesized to identify li-
gands to the Bz site on GABAA-R endowed with high affinity and/
or selective efficacy as promnemonic agents. All new compounds
were studied in in vitro tests and six compounds, with opportune
features in their substituents, were chosen for preliminary in vivo
evaluation of their potential effect on learning, memory and anxi-
ety. The modification (O/NH at position 8 and I/Me at position 3) on
lead compound 1 allowed us to obtain compounds 3 and 10 that
have a selective promnemonic effect at 1 and 3 mg/kg. The selec-
tivity of these two compounds was assessed in recombinant sub-
5. Experimental section
5.1. Chemistry
Melting points were determined with a Gallenkamp apparatus
and were uncorrected. Silica gel plates (Merk F254) and silica gel
types GABAA-R (a1b2c2, a2b2c2, a5b3c2.): compound 3 was able
Figure 3. Effect of compounds 3, 7, 10, 13, 14 and 22 in light–dark box test. All drugs were administered po 30 min before the test. Each value represents the mean of eight
mice. First column represents the number of transfer in 5 min, and the second column represents the time spent in light. ⁄P <0.01 versus control mice. §P <0.01 versus
flumazenil-treated mice.

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G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
Table 5
(5 mL) was added an excess of suitable amine (2 mL) and was kept
under vigorous stirring at 50 °C. TLC monitored the reaction and,
when the starting material disappeared, ice was added to obtain
a precipitate. Compounds 2 and 5 were purified by column chro-
matography (toluene/ethyl acetate 8/2)
Lack of anticonvulsant activity of 3, 7, 10, 13, 14, 22 in comparison with diazepam in
PTZ treated mice
Compounds Treatment (mg/kg)
po
% Protection on PTZ induced
convulsions
CMC 1%
Diazepam
3
0.1 mL
10
1
7.1
100*
0
5.1.2. 3-Iodo-8-(pyridin-4-ylmethylamino)pyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (2)
7
10
0
Yellow crystals; TLC eluent: toluene/ethyl acetate 8:2 v/v; IR
10
13
14
22
10
10
10
10
0
1
m
cmꢁ1 3271; H NMR (DMSO-d6) d 8.54 (d, 2H, H-20 and H-60,
10
10
0
J = 5.5 Hz); 8.37 (m, 1H, NH, exch.); 8.23 (s, 1H, H-2); 8.14 (d,
1H, H-6, J = 8.9 Hz); 7.38 (d, 2H, H-30 and H-50, J = 5.5 Hz); 7.03
(m, 2H, H-7 and H-9); 4.55 (d, 2H, CH2-N, J = 5.2 Hz). Anal. C,
H, N.
Pentylenetetrazole (PTZ) 90 mg kgꢁ1 ip. All compounds were administered 30 min
before test. Each value represents the mean of 10 mice.
P <0.01 versus control mice.
*
5.1.3. 3-Iodo-8-(pyridin-3-ylmethylamino)pyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (3)
Table 6
Affinity value at recombinant
Yellow crystals; TLC eluent: toluene/ethyl acetate 8:2 v/v; IR
m
a
1,2,5b2c2 GABAA/Bz complex subtypes
I%a or Kib (nM)
1
cmꢁ1 3270; H NMR (DMSO-d6) d 8.67 (s, 1H, H-20); 8.50 (d, 1H,
H-60, J = 4.9 Hz); 8.42 (t, 1H, NH, exch., J = 5.2 Hz); 8.25 (s, 1H, H-
2); 8.15 (d, 1H, H-6, J = 8.9 Hz); 7.80 (d, 1H, H-40, J = 7.8 Hz); 7.40
(dd, 1H, H-50, J = 7.8, 4.9 Hz); 7.05 (m, 2H, H-7 and H-9); 4.60 (d,
2H, CH2-N, J = 5.2 Hz). Anal. C, H, N.
No.
Cortex
a1
a2 (%)
a5
3
10
0.33 0.07
24.1 2.0
0.45 0.04
22.7 2.0
37
5
24.9 2.0
36%
a
Percent of inhibition of specific [3H]Ro15-1788 binding at 10
lM concentration
5.1.4. 3-Iodo-8-(pyridin-2-ylmethylamino)pyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (4)
are means SEM of five determinations.
b
Ki value are means SEM of five determinations.
Yellow crystals; TLC eluent: toluene/ethyl acetate 8:2 v/v; IR
m
1
cmꢁ1 3270; H NMR (DMSO-d6) d 8.58 (d, 1H, H-60, J = 5.0 Hz);
8.40 (t, 1H, NH, exch., J = 5.3 Hz); 8.23 (s, 1H, H-2); 8.12 (d, 1H,
H-6, J = 8.9 Hz); 7.79 (dt, 1H, H-40, J = 8.0, 2.1 Hz); 7.42 (d, 1H, H-
30, J = 8.0 Hz); 7.32 (dt, 1H, H-50, J = 7.7, 1.2 Hz); 7.06 (m, 2H, H-7
and H-9); 4.65 (d, 2H, CH2-N, J = 5.3 Hz). Anal. C, H, N.
60 (Merk 70–230 mesh) were used for analytical and column chro-
matography, respectively. The structures of all compounds were
supported by their IR spectra (KBr pellets in nujol mulls, Perkin–El-
mer 1420 spectrophotometer) and 1H NMR data (measured with a
Bruker 400 MHz). Chemical shifts were expressed in d ppm, using
DMSO-d6 or CDCl3 as solvent. The chemical and physical data of
new compounds are shown in Table 1; all new compounds possess
a purity P95%: microanalyses were performed with a Perkin–El-
mer 260 analyzer for C, H, N. The crystal structure of compound
25 was solved by means of single-crystal X-ray diffraction.
5.1.5. 3-Iodo-8-(fur-2-ylmethylamino)pyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (5)
Yellow crystals; TLC eluent: toluene/ethyl acetate 8:2v/v; IR
m
cmꢁ1 3272; 1H NMR (DMSO-d6) d 8.28 (s, 1H, H-2); 8.22 (t, 1H,
NH, exch., J = 5.3 Hz); 8.14 (d, 1H, H-6, J = 8.9 Hz); 7.65 (s, 1H, H-
50); 7.18 (d, 1H, H-9, J = 2.7 Hz); 7.03 (dd, 1H, H-7, J = 8.9, 2.7 Hz);
6.43 (m, 2H, H-30 and H-40); 4.55 (d, 2H, CH2-N, J = 5.3 Hz). Anal.
C, H, N.
5.1.1. General procedure for the synthesis of 2–5
To
the
starting
material
3-iodo-8-chloro
pyrazolo
[5,1-c][1,2,4]benzotriazine 5-oxide I42 (0.150 mmol) in EtOH
Figure 4. Release of ACh of 10. Saline and compound 10 were injected at time ‘0’, ip number of rat (Wistar): 6. ⁄P <0.01 versus saline.

G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
2195
5.1.6. 3-Iodo-8-(1-oxidopyridin-4-ylmethoxy)pyrazolo[5,1-
5.1.11. 3-Methyl-8-(2-chloropyridin-4-ylmethyloxy)pyrazolo
c][1,2,4]benzotriazine 5-oxide (6)
[5,1-c][1,2,4]benzotriazine 5-oxide (11)
An aqueous solution of hydrogen peroxide (30%, 3 mL) in acetic
acid (17 mL) was added to 141 (210 mg, 0.5 mmol) and the mixture
was stirred at 90 °C for 3 h. The mixture was cooled, and a second
portion of H2O2 (1 mL) was added. The mixture was stirred for an-
other 15 h at 90 °C monitoring by TLC (eluent: dichloromethane/
methanol 10:1 v/v). The pH was adjusted to 6 with aqueous solu-
tion of sodium hydroxide (10%). The yellow crude precipitate was
filtered off and was recrystallyzed by methoxyethanol. Orange
From 9 yellow crystals; TLC eluent: toluene/ethyl acetate 8:2: v/
1
v; H NMR (DMSO-d6) d 8.48 (d, 1H, H-60, J = 5.5 Hz)); 8.38 (d 1H,
H-6, J = 8.9 Hz); 8.14 (s, 1H, H-2); 7.76 (d, 1H, H-9, J = 2.7 Hz);
7.68 (s, 1H, H-30); 7.56 (d, 1H, H-50, J = 5.5 Hz); 7.38 (dd, 1H, H-7,
J = 8.9, 2.7 Hz); 5.50 (s, 2H, CH2-O); 2.26 (s, 3H, CH3). Anal. C, H, N.
5.1.12. 3-Methyl-8-(fur-2-ylmethyloxy)pyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (12)
crystals; IR
m
cmꢁ1 1538; 1H NMR (DMSO-d6) d 8.40 (d, 1H, H-6,
From 9 yellow crystals; TLC eluent: toluene/ethyl acetate 8:2 v/
v; 1H NMR (DMSO-d6) d 8.35 (d, 1H, H-6, J = 8.9 Hz); 8.15 (s, 1H, H-
2); 7.85 (d, 1H, H-9, J = 2.7 Hz); 7.75 (m, 1H, H-30); 7.30 (dd, 1H, H-
7, J = 8.9, 2.7 Hz); 6.70 (m, 1H, H-50); 6.53 (m, 1H, H-40); 5.40 (s, 2H,
CH2-O); 2.25(s, 3H, CH3). Anal. C, H, N.
J = 8.9 Hz); 8.38 (s, 1H, H-2); 8.27 (d, 2H, H-20 and H-60,
J = 5.5 Hz); 7.77 (d, 1H, H-9, J = 2.7 Hz); 7.55 (d, 2H, H-30 and H-
50, J = 5.5 Hz); 7.38 (dd, 1H, H-7, J = 8.9, 2.7 Hz); 5.45 (s, 2H,
CH2O). Anal. C, H, N.
5.1.7. 3-Methyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine (8)
To 1 mmol of compound II43 in dry THF (100 mL) a solution of
2.5 mmol borane dimetilsulphide complex (BMS) was added. The
temperature was maintained approximately at 0° during the addi-
tion. The solution was then brought to reflux and maintained there
for 1 h. Then, when the temperature was about 0 °C, the reaction
mixture was quenched by the slow addiction of 10 mL of methanol.
HCl conc. was then added slowly (pH 2), refluxed for 1 h and then,
when the temperature was 0 °C, sodium hydroxide solution (40%)
were added. The aqueous phase was extracted with ether (three
times with a total of 100 mL). After drying with sodium sulfate, a
precipitate was recovered. Yellow crystals; TLC eluent: toluene/
5.1.13. 3-(1,2,4-Oxadiazol-3-methyl-5-yl)-8-benzyloxypyrazolo
[5,1-c][1,2,4]benzotriazine5-oxide (13)
From V46 yellow crystals; TLC eluent: dichloromethane/metha-
nol 10:1 v/v; 1H NMR (DMSO-d6) d 8.91 (s, 1H, H-2); 8.44 (d, 1H, H-
6, J = 8.9 Hz); d 7.86 (d, 1H, H-9, J = 2.7 Hz); 7.55 (m, 2H, H20 and
H60); 7.44 (m, 4H, H-7, H-30, H-40 and H-50); 5.49 (s, 2H, CH2O);
2.43 (s, 3H, CH3). Anal. C, H, N.
5.1.14. 3-(1,2,4-Oxadiazol-3-methyl-5-yl)-8-(pyridin-4-
ylmethoxy)pyrazolo[5,1-c][1,2,4] benzotriazine5-oxide (14)
From V46 yellow crystals; TLC eluent: dichloromethane/metha-
nol 10:1 v/v; 1H NMR (DMSO-d6) d 8.92 (s, 1H, H-2); 8.67 (d, 2H, H-
20 and H-60, J = 5.5 Hz)); 8.47 (d, 1H, H-6, J = 8.9, 2.7 Hz); d 7.87 (d,
1H, H-9, J = 2.7 Hz); 7.61 (d, 2H, H-30 and H-50, J = 5.5 Hz)); 7.52
(dd, 1H, H-7, J = 8.9, 2.7 Hz); 5.61 (s, 2H, CH2O); 2.43 (s, 3H, CH3).
Anal. C, H, N.
ethyl acetate 8:2v/v; 1H NMR (CDCl3)
d 8.55 (d, 1H, H-6,
J = 8.9 Hz); 8.43 (d, 1H, H-9, J = 2.7 Hz); 8.09 (s, 1H, H-2); 7.67
(dd, 1H, H-7, J = 8.9, 2.7 Hz); 2.70 (s, 3H, CH3). Anal. C, H, N.
5.1.8. 3-Methyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide (9)
To a solution of 8 (0.1 mmol) in acetic acid (30 mL) were added
hydrogen peroxide 30% (6 mL).
The solution was refluxed for 8 h and the precipitate was fil-
tered and recrystallized obtaining the same product reported in
our previously paper.45
Yellow crystals; TLC eluent: isopropyl ether/cyclohexane 8:3 v/
v; 1H NMR (CDCl3) d 8.45 (d, 1H, H-6, J = 8.9 Hz); 8.35 (d, 1H, H-9,
J = 2.7 Hz); 7.90 (s, 1H, H-2); 7.55 (dd, 1H, H-7, J = 8.9, 2.7 Hz); 2.40
(s, 3H, CH3). Anal. C, H, N.
5.1.15. 3-(1,2,4-Oxadiazol-3-methyl-5-yl)-8-(thien-2-
ylmethoxy)pyrazolo[5,1-c][1,2,4]benzo triazine5-oxide (15)
From V46 yellow crystals; TLC eluent: dichloromethane/metha-
nol 10:1 v/v; 1H NMR (DMSO-d6) d 8.92 (s, 1H, H-2); 8.43 (d, 1H, H-
6, J = 8.9 Hz); d 7.91(d, 1H, H-9, J = 2.7 Hz); 7.63 (d, 1H, H-50,
J = 4.8 Hz); 7.45 (dd, 1H, H-7, J = 8.9, 2.7 Hz); 7.35 (d, 1H, H-30,
J = 3.5 Hz); 7.10 (dt, 1H, H-40, J = 4.8, 3.5 Hz); 5.70 (s, 2H, CH2-O);
2.44 (s, 3H, CH3). Anal. C, H, N.
5.1.16. 3-Carbamoyl-8-benzyloxypyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (16)
5.1.9. General procedure for the synthesis of 10–18
From III43 yellow crystals; TLC eluent: toluene/ethyl acetate 8:2
The reaction was carried out in 10 mL of dichloromethane to
which was added the starting material 9, III,43 V,46 (0.3 mmol),
5 mL of 40% sodium hydroxide solution, 0.5 mmol of tetrabutylam-
monium bromide (PTC), and the suitable alcohol in large excess
(ratio with starting material 1:15) under vigorous stirring. The
reaction was monitored by TLC, and when the starting material
disappeared, the organic layer was separated and the aqueous
layer extracted twice with 10 mL of dichloromethane. The com-
bined organic extracts were evaporated and the residue was recov-
ered with isopropil ether and then recrystallized by suitable
solvent.
v/v; IR m
cmꢁ1 3340, 3290, 1684; 1H NMR (DMSO-d6) d 8.56 (s, 1H,
H-2); 8.40 (d 1H, H-6, J = 8.9 Hz); 7.82 (d, 1H, H-9, J = 2.7 Hz); 7.58
(br s, 1H, exch., NH); 7.54 (m, 2H, H-20 and H-60); 7.42 (m, 4H, H-7,
H-30,H-40 and H-50); 7.12 (br s, 1H, exch., NH); 5.47 (s, 2H, CH2O).
Anal. C, H, N.
5.1.17. 3-Carbamoyl-8-(pyridin-4-ylmethoxy)pyrazolo[5,1-c]
[1,2,4]benzotriazine 5-oxide (17)
From III43 yellow crystals; TLC eluent: dichloromethane/metha-
nol 10:1 v/v; IR
m
cmꢁ1 3347, 3200, 1685; 1H NMR (DMSO-d6) d
8.63 (d, 2H, H-20 and H-60, J = 5.5 Hz); 8.57 (s, 1H, H-2); 8.43 (d
1H, H-6, J = 8.9 Hz); 7.82 (d, 1H, H-9, J = 2.7 Hz); 7.58 (br s, 1H,
exch., NH); 7.53 (d, 2H, H-30 and H-50, J = 5.5 Hz); 7.47 (dd, 1H,
H-7, J = 8.9, 2.7 Hz); 7.12 (br s, 1H, exch., NH); 5.56 (s, 2H,
CH2-O). Anal. C, H, N.
5.1.10. 3-Methyl-8-(pyridin-4-ylmethyloxy)pyrazolo[5,1-c]
[1,2,4]benzotriazine 5-oxide (10)
From 9 yellow crystals; TLC eluent: dichloromethane/methanol
1
10:1 v/v; H NMR (DMSO-d6) d 8.62 (d, 2H, H-20 and H-60,
J = 5.5 Hz)); 8.38 (d, 1H, H-6, J = 8.9 Hz); 8.14 (s, 1H, H-2); 7.74
(d, 1H, H-9, J = 2.7 Hz); 7.52 (d, 2H, H-30 and H-50, J = 5.5 Hz);
7.36 (dd, 1H, H-7, J = 8.9, 2.7 Hz); 5.52 (s, 2H, CH2-O); 2.26 (s, 3H,
CH3). Anal. C, H, N.
5.1.18. 3-Carbamoyl-8-(thien-2-ylmethoxy)pyrazolo[5,1-c]
[1,2,4]benzotriazine 5-oxide (18)
From III43 yellow crystals; TLC eluent: toluene/ethyl acetate 8:2
v/v; IR
m
cmꢁ1 3445, 3315, 1670; 1H NMR (DMSO-d6) d 8.58 (s, 1H,

2196
G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
H-2); 8.39 (d, 1H, H-6, J = 8.9 Hz); 7.87 (d, 1H, H-9, J = 2.7 Hz); 7.63
(d, 1H, H-50, J = 4.8 Hz)); 7.55 (br s, 1H, exch., NH); 7.42 (dd, 1H, H-
7, J = 8.9, 2.7 Hz); 7.39 (d, 1H, H-30, J = 3.5 Hz); 7.09 (m, 2H, exch.,
NH and H-40); 5.56 (s, 2H, CH2-O). Anal. C, H, N.
5.1.26. 3-(1,2,4-Triazol-1-methyl-5-yl)-8-benzyloxypyrazolo
[5,1-c][1,2,4]benzotriazine 5-oxide (24)
To a suspension of 19 (0.30 mmol) in AcOH (10 mL) was added
methylhydrazine (0.05 mL). The reaction was kept at 90–100 °C for
2 h. The residue was filtered and recrystallized from a suitable sol-
vent. Yellow crystals; TLC eluent: dichloromethane/methanol 10:1
v/v; 1H NMR (CDCl3) d 8.50 (m, 2H, H-2 and H-6); 8.01 (s, 1H, CH
triazole); 7.88 (d, 1H, H-9, J = 2.7 Hz); 7.46 (m, 5H, phenyl); 7.31
(dd, 1H, H-7, J = 8.9, 2.7 Hz); 5.36 (s, 2H, CH2O); 4.16 (s, 3H, N-
CH3); Crystal data for compound 24 are reported in the Supple-
mentary data; Anal. C, H, N.
5.1.19. General procedure for the synthesis of 19–21
To compounds 16–18 (1.9 mmol) in anhydrous toluene
(10 mL) and anhydrous DMF (1 mL) was added dimethylformam-
ide–dimethylacetale (DMF–DMA) to obtain the intermediates
19–21. The reaction mixture was stirred at 100–110 °C for 2 h
and after cooling, the residue was filtered and washed with
diethylether.
5.1.27. 3-(1,2,4-Triazol-1-methyl-3-yl)-8-benzyloxypyrazolo
[5,1-c][1,2,4]benzotriazine 5-oxide (25)
5.1.20. 3-(N-(Dimethylaminomethylene)carbamoyl)-8-
benzyloxypyrazolo[5,1-c][1,2,4] benzotriazine 5-oxide (19)
From 16 yellow crystals; TLC eluent: dichloromethane/metha-
Method a: To a solution of (0.35 mmol) 22 in anhydrous DMF
(15 mL) was added
a suspension of 50% NaH (0.76 mmol,
nol 10:1 v/v; IR
m
cmꢁ1 1638; 1H NMR (DMSO-d6) d 8.63 (s, 1H,
18.2 mg) and an excess of CH3I (0.05 mL).The reaction was kept
at 50–60 °C for 18 h. The final solution was evaporated and the
mixture of two isomers (3:1) was purified by recrystallization with
ethanol obtaining compound 25.
Method b: A regioselective alkylation was realized using LiOH
as base and 25 as pure product was obtained. To a solution of 22
(0.035 mmol) in EtOH 96% (15 mL) was added a solution of LiOH
0.25 M (5 mL) and CH3I (0.035 mL). Then the reaction mixture
was refluxed for 20 h and the formed precipitate was filtered and
purified by recrystallization.
Yellow crystals; TLC eluent: dichloromethane/methanol 10:1 v/
v; 1H NMR (CDCl3) d 8.62 (s, 1H, H-2); 8.50 (d, 1H, H-6, J = 8.9 Hz);
8.14 (s, 1H, CH triazole); 7.85 (d, 1H, H-9, J = 2.7 Hz); 7.46 (m, 5H,
phenyl); 7.24 (dd, 1H, H-7, J = 8.9, 2.7 Hz); 5.33 (s, 2H, CH2O); 4.05
(s, 3H, N-CH3). Anal. C, H, N.
H-2); 8.61 (s, 1H, @CH); 8.38 (d 1H, H-6, J = 8.9 Hz); 7.82 (d, 1H,
H-9, J = 2.7 Hz); 7.54 (m, 2H, H-20 and H-60); 7.42 (m, 4H, H-7, H-
30, H-40 and H-50); 5.46 (s, 2H, CH2O); 3.20 (s, 3H, N-CH3); 3.17
(s, 3H, N-CH3). Anal. C, H, N.
5.1.21. 3-(N-(Dimethylaminomethylene)carbamoyl)-8-(pyridin-
4-ylmethoxy)pirazolo[5,1-c][1,2,4]benzotriazine 5-oxide (20)
From 17 yellow crystals; TLC eluent: dichloromethane/metha-
nol 10:1 v/v; IR
m
cmꢁ1 1641; 1H NMR (DMSO-d6) d 8.62 (m, 4H,
H-20, H-60; H-2 and @CH); 8.41 (d, 1H, H-6, J = 8.9 Hz); 7.81 (d,
1H, H-9, J = 2.7 Hz); 7.54 (m, 2H, H-30 and H-50); 7.44 (dd, 1H, H-
7, J = 8.9, 2.7 Hz); 5.55 (s, 2H, CH2-O); 3.21 (s, 3H, N-CH3); 3.17
(s, 3H, N-CH3). Anal. C, H, N.
5.1.22. 3-(N-(Dimethylaminomethylene)carbamoyl)-8-(thien-2-
ylmethoxy)pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (21)
From 18 yellow crystals; TLC eluent: dichloromethane/metha-
5.2. Radioligand binding assay on bovine cerebral cortex
membranes and on GABAA receptor subtypes
nol 10:1 v/v; IR
m
cmꢁ1 1640; 1H NMR (DMSO-d6) d 8.64 (s, 1H,
H-2); 8.60 (s, 1H, @CH); 8.38 (d, 1H, H-6, J = 8.9 Hz); 7.86 (d, 1H,
H-9, J = 2.7 Hz); 7.62 (d, 1H, H-50, J = 4.8 Hz); 7.38 (dd, 1H, H-7,
J = 8.9, 2.7 Hz); 7.34 (d, 1H, H-30, J = 3.5 Hz); 7.09 (dt, 1H, H-40,
J = 4.8, 3.5 Hz); 5.66 (s, 2H, CH2-O); 3.20 (s, 3H, N-CH3); 3.19 (s,
3H, N-CH3). Anal. C, H, N.
[3H]Ro15-1788 (specific activity 70.8 Ci/mmol) was obtained
from NEN Life Sciences products. All the other chemicals, which
were of reagent grade, were obtained from commercial suppliers.
Bovine cerebral cortex membranes were prepared as previously
described.51 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.52
[3H]Ro15-1788 binding studies were performed as previously re-
ported. Clonal mammalian cell lines, expressing relatively high lev-
5.1.23. General procedure for the synthesis of 22–23
To a suspension of 19 (0.30 mmol) in AcOH (10 mL) was added
hydrazine hydrate (0.30 mmol, 0.05 mL). The reaction was kept at
90–100 °C for 2 h. The residue was filtered and recrystallized from
a suitable solvent.
els of GABAA receptor subtypes (
maintained as previously described in minimum essential medium
Eagle’s with EBSS, supplemented with 10% fetal calf serum, -gluta-
mine (2 mM), penicillin (100 U/ml), and streptomycin (100 g/ml)
a1b2c2, a2b2c2, a5b3c2), were
L
5.1.24. 3-(1,2,4-Triazol-3-yl)-8-benzyloxypyrazolo[5,1-c][1,2,4]
benzotriazine 5-oxide (22)
l
in a humidified atmosphere of 5% CO2/95% air at 37 °C. After re-
moval, the cells were harvested by centrifugation at 500ꢂg. The
crude membranes were prepared after homogenization in 10 mM
potassium phosphate, pH 7.4, and differential centrifugation at
48,000 g for 30 min at 4 °C. The pellets were washed twice in this
manner before final resuspension in 10 mM potassium phosphate,
pH 7.4, that contained 100 mM potassium chloride [3H]Ro15-1788
binding assays to transfected cell membranes was carried out as
previously described.
From 19 yellow crystals; TLC eluent: toluene/ethyl acetate 8:2
v/v; IR
m
cmꢁ1 3230; 1H NMR (DMSO-d6) d 11.7 (br s, 1H, exch.,
NH); 8.66 (s, 1H, H-2); 8.50 (s, 1H, CH triazole); 8.40 (d, 1H, H-6,
J = 8.9 Hz); 7.83 (d, 1H, H-9, J = 2.7 Hz); 7.55(m, 2H, H-20 and H-
60); 7.42 (m, 4H, H-7, H-30, H-40 and H-50); 5.47 (s, 2H, CH2O). Anal.
C, H, N.
5.1.25. 3-(1,2,4-Triazol-3-yl)-8-(pyridin-4-ylmethoxy)
pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (23)
In brief, the cell line membranes were incubated in a volume of
From 20 yellow crystals; TLC eluent: dichloromethane/metha-
500 l
l, which contained [3H]Ro15-1788 at a concentration of 1–
nol 10:1 v/v; IR
m
cmꢁ1 3390; 1H NMR (DMSO-d6) d 11.9 (br s,
2 nM and test compound in the 10ꢁ9–10ꢁ5 M range. Nonspecific
binding was defined by 10ꢁ5 M diazepam. Assays were incubated
to equilibrium for 1 h at 4 °C. The compounds were dissolved in
DMSO, the level of which did not exceed 1% and which was main-
tained constant in all tubes. At least six different concentrations of
1H, exch., NH); 8.63 (m, 4H, H-20, H-60; H-2 and CH triazole);
8.43 (d 1H, H-6, J = 8.9 Hz); 7.83 (d, 1H, H-9, J = 2.7 Hz); 7.53 (d,
2H, H-30 and H-50, J = 5.5 Hz); 7.43 (dd, 1H, H-7, J = 8.9, 2.7 Hz);
5.56 (s, 2H, CH2-O). Anal. C, H, N.

G. Guerrini et al. / Bioorg. Med. Chem. 21 (2013) 2186–2198
2197
each compound were used. The data of n = 5 experiments carried
out in duplicate were analyzed by means of an iterative curve-fit-
ting procedure (program Prism, GraphPad, San Diego, CA), which
provided IC50, Ki, and SEM values for tested compounds, the Ki val-
ues being calculated from the Cheng and Prusoff equation.53
5.3.6. Determination of ACh release by cerebral microdialysis
Microdialysis was performed in the rat parietal cortex according
to Giovannini.56 The coordinates used for implantation of the hor-
izontal microdialysis tube (AN 69 membrane, molecular weight cut
off >15 kDa, Dasco, Italy) were AP 0.5 mm and H 2.3 mm from the
bregma.57 One day after surgery, the microdialysis tube was per-
fused at a constant flow rate (2 pl/min) with Ringer solution (NaCl
147, KCl 4.0, CaCl2 1.2 mM) containing 7 pM physostigmine sul-
phate. After a 1 h settling period, the perfusate was collected at
20 min intervals in test tubes containing 5 pl of 0.5 mM HC1 to pre-
vent ACh hydrolysis. The samples were then assayed for ACh con-
tent, either immediately or after freezing. ACh was detected and
quantified by high performance liquid chromatography (HPLC)
with an electrochemical detector, as described by Damsma.58
5.3. Pharmacological methods
The experiments were carried out in accordance with the Ani-
mal 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 suf-
fering and to reduce the number of animals involved. Male CD-1 al-
bino 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
12h light/dark cycles (lights on at 7 am). The cages were brought
into the experimental room the day before the experiment, for
acclimatization purposes. All experiments were performed be-
tween 10:00 am and 15:00 pm.
5.4. Drugs
Diazepam (Valium 10—Roche), flumazenil (Roche), pentylen-
etetrazole (PTZ) (Sigma), scopolamine (Sigma) were used. All drugs
except PTZ were suspended in 1% carboxymethylcellulose sodium
salt and sonicated immediately before use. PTZ was dissolved in
isotonic (NaCl 0.9%) saline solution. Drug concentrations were pre-
pared in such a way that the necessary dose could be administered
in a volume of 10 ml/kg by the po, ip or sc routes.
5.3.1. Passive-avoidance test
The test was performed according to the step-through method
described by Jarvik.54 The apparatus consisted of a two-compart-
ment acrylic box with a lighted compartment connected to a dark-
ened one by a guillotine door. As soon as the mouse entered the
dark compartment, it received 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.
5.5. Statistical analysis
Results are given as the mean SEM. Statistical analysis was
performed by means of ANOVA, followed by Scheffe’s post-hoc
test. Student’s two-tailed t-test was used to verify the significance
between two means. Data were analyzed using a computer pro-
gram (Number Cruncher Statistical System, Version 5.03 9/92).
For percentage values, Chi-Square analysis was used in accordance
with Tallarida and Murray. P <0.05 were considered significant.
5.3.2. Social learning test
The social learning test was performed according to Dantzer,55
Juvenile male rats (80–100 g) were used as social stimuli for study-
ing adult rats (220–240 g). On the first day of the experiment, a
juvenile rat was introduced into the adult male’s cage and the time
spent in social-investigatory behaviour by the adult male within a
5 min fixed interval was recorded. After 24 h, the same juvenile rat
was placed again into the adult male’s cage and social-investiga-
tory behaviour was recorded during a 5 min interval. On the same
day, the social-investigatory behaviour towards a second unfamil-
iar juvenile rat was also recorded.
5.6. Crystallographic analyses
The data were collected at 150(2)K on Xcalibur3 CCD 4-circle
diffractometer using a graphite monochromator, Mo Ka radiation.
A reference frame was monitored every 50 frames to control the
stability of the crystal and the system revealed no intensity decay.
The data set was corrected for Lorentz, polarization effects, absorp-
tion corrections were performed by the ABSPACK multiscan proce-
dure of the CrysAlis59 data reduction package. The structure was
solved using direct method with SHELXS-97 software, and the
refinement was carried out using the SHELXL-9760 software pack-
age. All non-hydrogen atoms were located from the initial solution
or from subsequent electron density difference maps during the
initial course of the refinement. After locating the non-hydrogen
atoms, the models were refined against F2, first using isotropic
and finally anisotropic thermal displacement parameters. Finally,
the hydrogen atoms were located from the electron density differ-
ence maps and isotropically refined. Programs used in the crystal-
lographic calculations included WinGX61 and ORTEP for graphics.62
Crystal structural data are available from the Cambridge Crystallo-
graphic Data Center CCDC No. 846436
5.3.3. Mouse light/dark box test
The apparatus (50 cm long, 20 cm wide, and 20 cm high) con-
sisted of two equal acrylic compartments, one dark and one
light, illuminated by a 60 W bulb lamp and separated by a divi-
der with a 10 ꢂ 3-cm opening at floor level. Each mouse was
tested by placing it in the center of the lighted area, facing away
from the dark one, and allowing it to explore the novel environ-
ment for 5 min. The number of transfers from one compartment
to the other and the time spent in the illuminated side were
measured. This test exploited the conflict between the animal’s
tendency to explore a new environment and its fear of bright
light.
5.3.4. 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 24 rpm. The number of falls from the rod in 30 s were
counted 15, 30, 45 and 60 min after drug administration.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.027.
5.3.5. Pentylenetetrazole (PTZ)-induced seizure
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