Development of ligands at γ-aminobutyrric acid type A (GABA A) receptor subtype as new agents for pain relief

Article abstract of DOI:10.1016/j.bmc.2011.10.047

The identification of compounds with selective anxiolytic-like effects, exerted through the benzodiazepine site on γ-aminobutyric acid type A (GABA_A) receptors, and that show pronounced antihyperalgesia in several pain models, has oriented rese

Full text of DOI:10.1016/j.bmc.2011.10.047

Bioorganic & Medicinal Chemistry 19 (2011) 7441–7452
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of ligands at
c-aminobutyrric acid type A (GABAA) receptor
subtype as new agents for pain relief
Gabriella Guerrini a, , Giovanna Ciciani a, Fabrizio Bruni a, Silvia Selleri a, Claudia Martini b,
⇑
Simona Daniele c, Carla Ghelardini d, Lorenzo Di Cesare Mannelli d, Annarella Costanzo a
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, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università degli Studi di Pisa, via Bonanno, 6, 56126 Pisa, Italy
c Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, Italy
d Dipartimento di Farmacologia Preclinica e Clinica Aiazzi-Mancini, Università degli Studi di Firenze, Viale Pieraccini, 6, 50139 Firenze, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The identification of compounds with selective anxiolytic-like effects, exerted through the benzodiaze-
Received 31 August 2011
Revised 7 October 2011
Accepted 14 October 2011
Available online 21 October 2011
pine site on c-aminobutyric acid type A (GABAA) receptors, and that show pronounced antihyperalgesia
in several pain models, has oriented research towards the development of new agents for the relief of
pain. Starting from our previously reported ligands at the benzodiazepine site on GABAA receptors show-
ing selective anxiolytic-like effects, we have designed new compounds with the aim of identifying those
devoid of the typical side effects of the classical benzodiazepines. Our preliminary results indicate that
compounds 4, 10( ) and 11 have a very promising antihyperalgesic profile in different animal pain mod-
els (peripheral mono-neuropathy, STZ-induced hyperalgesia). In particular 11 exhibits high potency since
it exerted its protective effect starting from the dose of 3 mg/kg po, after single injection.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Pyrazolo[51-c][1,2,4]benzotriazine system
GABAA receptors
Binding affinity
Antihyperalgesic effect
Anxiolytic-like effect
1. Introduction
on GABAA-Rs (otherwise called ligands to GABAA-Rs subtype), situ-
ated at the interface between the alpha and gamma subunits, the
The importance of the
c
-aminobutyrric acid (GABA) neuro-
conformational changes can produce either the potentiation (ago-
nist or PAM, positive allosteric modulator) or the decrease (inverse
agonist or NAM, negative allosteric modulator) in the response
elicited by GABA or no event (antagonist).2–4
transmitter in mediating or modulating of many functions of the
central nervous system cannot be questioned. It is involved in
many physiological functions related to its key role in the fast syn-
aptic inhibition in the brain and spinal cord via activation of synap-
tic or extrasynaptic ionotropic GABA receptors (GABAA-receptors).
Deficits in the functional expression of these receptors are critical
in a large number of human neurological and psychiatric diseases
such as epilepsy, anxiety disorder, cognitive deficits, schizophrenia,
depression and pain.1
It is known that GABAA-Rs are extensively distributed within
peripheral and spinal nociceptive system5 and play an important
role in the mediation of pain transmission. Although a great variety
of mediators are involved in pain pathologies, the loss of synaptic
inhibition of glycinergic and/or GABAergic input after nerve injury
or tissue trauma or inflammation (becoming less inhibitory or even
excitatory) has been recognized as an important process in the
development and maintenance of pain of various origin.6 Even if
the precise basis of this dis-inhibition may be attributed to different
causes (loss of GABAergic interneurons, reduced GABA storage/re-
lease, alteration of cation-chloride co-transporter),7,8 it is clear that
restoring spinal synaptic inhibition, through pharmacological facil-
itation of GABAA-Rs, is an interesting and intriguing approach for
the treatment of pain.9,10 Many authors have reported that benzo-
diazepines (mainly midazolam) modulate pain perception both in
animal experiments11–13 and in human patients14,15 even though
the complexity of the intrathecal administration precludes routine
use. On the other hand, even though systemic administration of
GABAA-R is a pentameric arrangement of different subunits (
, d, , h, and ), many of which exist in multiple isoforms (
1–6, b 1–3, 1–3, 1–3), with a typical composition of 2 , 2b
and 1 or 1d Activation of GABAA-R causes an influx of chloride
a,
a
b,
c
e
p
q
c
q
a
c
ion and hyperpolarization of the neuron. The allosteric modulation
of GABAA-Rs by benzodiazepine and/or non-benzodiazepine
ligands induces conformational changes in the multi-subunit
receptor that open the central channel and the chloride ions move
into the cells. Especially for the ligands at the benzodiazepine site
⇑
Corresponding author. Tel. +39 055 4573766; fax: +39 055 4573671.
E-mail address: gabriella.guerrini@unifi.it (G. Guerrini).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.047

7442
G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
midazolam can induce antihyperalgesic action in the formalin
test,16 this effect can be masked or complicated by the sedative ef-
fects, thus reducing its use against pain.
neuropathy, and in the mouse abdominal constriction test follow-
ing injection of acetic acid 0.6%.
Benzodiazepine-sensitive GABAA-Rs subtypes contain four
types of alpha isoform (a1–3 and a5), to which it has been possible
2. Chemistry
to attribute the effects on sedation, anxiety and memory of diaze-
pam (full agonist), using GABAA-Rs point-mutated knock-in
mice.17–23 Using this type of approach it has recently been reported
The 3-methyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide,
3
and the previous synthesized 3-iodo-8-chloropyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide34 were used as starting material for the
synthesis of the corresponding 3-methyl-(compounds 4–8) and 3-
iodo-(compounds 11–15), either 8-arylalkyloxy- or 8-arylalkylamino
derivatives.
that the GABAA-Rs containing
fect) are the principal contributors (and to a lesser extent -
a
2 (in addition to the anxiolytic ef-
3) to
a
the antihyperalgesic actions of classical benzodiazepines (e.g.,
diazepam) administered intrathecally24 or systemically.25 This ef-
fect is evidenced when the pain sensitivity is increased pathologi-
cally (inflammation or neuropathic injury) in agreement with the
notion that the benzodiazepines are generally not analgesic per
Compound 3, Scheme 1, was obtained by exploiting the conden-
sation between the 2-methyl-3-oxo-propanenitrile sodium salt
(sodium 2-cyanoprop-1-en-1-olate) and 2-nitro-5-chlorophenyl-
hydrazine in ethanol solution at pH 1 (with hydrochloric acid).
During the reaction was evidenced the formation of two products
that, identified step by step, resulted to be the 1-(2-nitro-5-chloro-
se. Thus, the use of
a2 selective agonist could be a useful tool for
treatment of pain disorders as reported in several rodent pain
models, in which pronounced antihyperalgesia was obtained,25–28
without sedative effects.
phenyl)-2H-4-methyl-5-iminopyrazole
1 and the 1-(2-nitro-
5-chlorophenyl)-4-methyl-5-aminopyrazole 2. Both the products
cyclized to 3-methyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide, 3 when reacted in 10% sodium hydroxide solution (see
Scheme 1).
The 8-arylalkyloxy derivatives 4, 5, 6( ) were obtained by
exploiting the nucleophilic substitution of the chlorine atom at po-
sition 8 of the starting materials 3, by using the suitable alcohols,
with phase transfer catalyst (PTC). The 8-arylalkylamino deriva-
tives, 7–8, 11–15, were instead obtained by treating the starting
materials 3 and the 3-iodo-8-chloropyrazolo[5,1-c][1,2,4]benzotri-
azine 5-oxide,34 with an excess of suitable amine and easily recov-
ered as the principal compound at the end of the reaction,
Scheme 2.
Recently we have reported that some derivatives with the pyr-
azolo[5,1-c][1,2,4]benzotriazine 5-oxide core have selective anxio-
lytic-like properties29–32 without exhibiting sedative and
myorelaxant effects. Since the relationship between anxiolysis
and antihyperalgesia is intriguing we decided to investigate the
antihyperalgesic profile of some compounds we have already re-
ported as selective anxiolytics (I,29 II,32 and III,30 see Chart 1).
Based on the pharmacological results of these compounds (data
reported in the Supplementary data) the derivative with the most
promising antihyperalgesic effects was III,30 thus we synthesized
new 3-iodio- and 3-methyl-, 8-aryalkyloxy- and/or 8-arylalkyl
aminopyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide derivatives, as
its optimization. Moreover, to better find out the features for anti-
hyperalgesic activity, we decided to test two already reported33,30
3-iododerivatives in vivo, in this work compounds 9 and 10( ).
Clearly, to assess the involvement of GABAA-Rs, the binding affinity
and the potential anxiolytic-like effects were compared with those
of diazepam and antagonized by flumazenil (GABAA-R antagonist).
Compounds having anxiolytic-like activity were then tested in
adult rats on a pain model in which peripheral mono-neuropathy
was produced; in the animal model of streptozotocin (STZ)-in-
duced hyperalgesia, which mimics the pain caused by diabetic
Compounds 4, 7 and 11 were deoxidized at position 5 by treat-
ment with zinc in acetic acid, obtaining respectively derivatives 4R,
7R and 11R, useful for study of SAR, Scheme 3. All compounds de-
scribed here are listed in Table 1.
3. Results and discussion
The Bz site/GABAA-R binding affinity of newly synthesized com-
pounds was evaluated by their ability to displace [3H]flumazenil
(Ro15–1788) from its specific binding in bovine brain membrane
N
Cl
N
O
Ia
S
S
O
N
N+
O-
Ki = 6.8 nM
Anxiolytic-like effect at 1-100 mg/kg
N
O
N
IIb
Ki = 8.9 nM
O
N
N+
O-
Anxiolytic-like effect at 10-30 mg/kg
N
O
N
IIIc
I
Ki = 8.9 nM
N
N+
O-
Anxiolytic-like effect at 3-30 mg/kg
Chart 1. see Ref.29 bsee Ref.32 csee Ref.30
; ;

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
7443
H3C
H
NH2
N
N
NH
Cl
Cl
NH
Cl
N
N
CH3
+
CH3
NH
NH2
ONa
NO2
a
b
NO2
NO2
1
2
c
N
Cl
N
CH3
N
N+
O-
3
Scheme 1. Reagents and conditions: (a) Ethanol /HCl, 80 °C 2 h; (b) Ethanol /HCl, 80 °C 10 h; (c) NaOH 10%, 50 °C.
N
N
Cl
N
RX
N
R3
a
R3
N
N+
O-
N
N+
O-
Comp
4
X
O
R
R3
CH2Ph
CH3
R3 = CH3
R3 = I
3
see ref.34
5
O
CH2-o-OCH3Ph CH3
6( )
7
O
CH2(CH)CH3Ph CH3
NH
CH2Ph
CH3
8
NH CH2-o-OCH3Ph CH3
11
12
13
14
NH
CH2Ph
I
I
I
I
I
NH CH2-o-OCH3Ph
NH
NH
CH2-p-ClPh
CH2CH2Ph
CH2Ph
15 NCH3
Scheme 2. Reagents: (a) ROH/NaOH 40% solution/NBu+BrÀ/CH2Cl2 for compounds 4–6( ); RNH2/EtOH for compounds 7–8, 11–15.
N
N
RX
N
R3
RX
N
a
R3
N
N+
O-
N
N
Comp
X
O
R
R3
CH3
CH3
I
Comp
X
O
R
R3
CH3
CH3
I
4
7
CH2Ph
CH2Ph
CH2Ph
4R
7R
CH2Ph
CH2Ph
CH2Ph
NH
NH
NH
NH
11
11R
Scheme 3. Reagents: (a) Acetic acid/zinc dust

7444
G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
Table 1
Chemical data for new synthesized compounds
N
N
N
H
N
N
R8
R8
N
R3
R3
Cl
Cl
N
N
CH3
CH3
N
N
N+
O-
N
NH
NH2
NO2
NO2
4R, 7R, 11R
1
2
3-15
Compd
R3
R8
Formula (MW)
Mp °C (recryst. solvent)
1
2
—
—
—
—
C
C
10H9N4O2Cl (252.66)
10H9N4O2Cl (252.66)
176–179
247–248 (Ethanol)
246–247 (Ethanol)
207–208 (Ethanol)
236–237 (Ethanol)
247–249 (Methoxyethanol)
133–134 (Ethanol)
>300 (Chromathography)
236–237 (Ethanol)
254–255 (Chromathography)
3
4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
I
I
I
I
I
I
I
I
Cl
C10H7N4OCl (234.64)
C17H14N4O2 (306.32)
C17H14N4O (290.31)
C18H16N4O3 (336.34)
C19H18N4O2 (334.38)
C17H15N5O (305.33)
C17H15N5 (289.33)
OCH2-Ph
OCH2-Ph
OCH2-o-OCH3-Ph
OCH2CH(CH3)-Ph
NHCH2-Ph
NHCH2-Ph
NHCH2-o-OCH3-Ph
O(CH2)4CH3
OCH2CH(CH3)-Ph
NHCH2-Ph
NHCH2-Ph
NHCH2-o-OCH3-Ph
NHCH2-p-Cl-Ph
NHCH2CH2-Ph
N(CH3) CH2-Ph
4Ra
5
6( )
7
7Ra
8
C18H17N5O2 (335.36)
9b
10( )c
11
11Ra
12
13
14
15
C16H12N5OI (417.20)
C16H12N5I (401.20)
C17H14N5O2I (447.23)
C16H11N5OICl (451.65)
C17H14N5OI (431.23)
C17H14N5OI (431.23)
239–240 (Methoxyethanol)
242–243 (Methoxyethanol)
238–239 (Ethanol)
>300 (Methoxyethanol)
240–241 (Ethanol)
221–223 (Methoxyethanol)
a
b
c
5-Deoxide derivatives.
See Ref. 33.
See Ref. 30.
Table 2
and was expressed as Ki value only for those compounds inhibiting
radioligand binding by more than 80% at fixed concentrations of
Binding data of new compounds
N
10 lM. The binding data (Table 2) show that all compounds were
R8
N
R3
able to bind to the Bz site/GABAA-R with good affinity, reaching,
for some derivatives 8, 11, 11R and 12, the subnanomolar range
(0.78 PKi (nM) P0.109).
N
N+
Compounds 3, 4, 5, 6( ), endowed with a 3-methyl group on the
pyrazolobenzotriazine 5-oxide system, show affinity in the range
of 400 Ki (nM) P3.02. The importance of the oxygen atom at posi-
tion 830,33 is evidenced by the weak binding affinity of compound 3
(Ki = 400 nM), the 8-chloro derivative, lacking of 8-oxygen atom.
Moreover also the derivative 8-(2-methyl-2-phenylethoxy)-, 6( ),
probably because of a steric hindrance of the branched 8-O-benzyl
chain (Ki = 361.2 nM), has weak affinity. These compounds have
lower affinity than the corresponding 3-iodo derivatives (8-chloro,
Ki = 22.8 nM and 8-(2-methyl-2-phenylethoxy)- Ki = 160 nM
respectively,30). This fact is probably due to reduced ability of the
methyl group to form a lipophilic interaction with the receptor
protein, as also indicated by the different value of the lipophilic
-
O
Compd
R3
R8
Cl
Kia (nM)
3
4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
I
I
I
I
I
I
I
I
400 11.0
4.66 0.02
1.60 0.07
3.02 0.2
361.2 25.6
2.58 0.13
2.78 0.06
0.78 0.04
6.5 0.4
160 15.0
0.109 0.008
0.71 0.07
0.138 0.005
2.44 0.1
OCH2-Ph
OCH2-Ph
OCH2-o-OCH3-Ph
OCH2CH(CH3)-Ph
NHCH2-Ph
NHCH2-Ph
NHCH2-o-OCH3-Ph
O(CH2)4CH3
OCH2CH(CH3)-Ph
NHCH2-Ph
NHCH2-Ph
NHCH2-o-OCH3-Ph
NHCH2-p-Cl-Ph
NHCH2CH2-Ph
N(CH3) CH2-Ph
4Rb
5
6( )
7
7Rb
8
9c
10( )d
11
11Rb
12
13
14
15
parameter,
p: methyl, p = 0.56 and iodine, p
= 1.12,.35 The 3-
methyl derivatives (4 and 5) show good affinity values (4,
Ki = 4.66 nM and 5, Ki = 3.02 nM) even if reduced by about 4–7-fold
compared to the corresponding 3-iodo derivatives (8-benzyloxy-
Ki = 1.1 nM and 8-o-methoxybenzyloxy- Ki = 0.42 nM30), confirm-
ing the importance of more lipophilic substituent at position 3.
When the position 8 was substituted with a moiety that con-
tains a hydrogen bond donor atom (–NH–), in the 3-methyl deriv-
atives (7–8) the affinity values improved 2–4-fold compared to the
corresponding 8-O-derivatives (4–5). Compare compound 7,
Ki = 2.58 nM with 4, Ki = 4.66 nM and compound 8, Ki = 0.78 nM
with 5, Ki = 3.02 nM, suggesting a positive influence of the NH
group.
14.72 1.0
1.02 0.10
a
b
c
Ki are means SEM of five determinations.
5-Deoxide derivatives.
See ref.33.
d
See Ref. 30.
a more lipophilic substituent, that is, iodine, compounds 11, 12
and 13 emerge with very good affinity value: 11, Ki = 0.109 nM;
12, Ki = 0.138 nM; 13, Ki = 2.44 nM. These results confirm the
importance of the lipophilic feature of the substituent at position
3 and, again, the key role of the hydrogen bond donor moiety (8-
NH- group) to better receptor recognition. The modification of
On the other hand, when the position
3 of the 8-aryl-
alkylaminopyrazolobenzotriazine core was again substituted with

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
7445
the 8-NH-CH2phenyl chain of compound 11 (Ki = 0.109 nM) in a
linear manner, (14, 8-NH-CH2CH2phenyl–) or in a branched man-
ner, (15, (8-N(CH3)CH2phenyl–) caused a 10–140-fold decrease in
affinity although always in the nanomolar range: 14, Ki = 14.72 nM
and 15, Ki = 1.02 nM. Thus the better influence of the 8-NH-
CH2phenyl group, even if modified, on receptor interaction than
the 8-O-benzyl group30 is evidenced; in fact, the affinity values of
the 8-NH- derivatives are 3–10-fold higher than the 8-O-deriva-
tives, as evidenced in a previous paper on derivatives bearing the
NH-pyrrole moiety.36
Interesting information was obtained from the 5-N-deoxide
derivatives 4R, 7R and 11R. These compounds have comparable
or better affinity values than their corresponding 5-oxide deriva-
tives. Compare 4R, Ki = 1.60 nM nM versus 4, Ki = 4.66 nM, 7R,
Ki = 2.78 nM versus 7, Ki = 2.58 nM and 11R, Ki = 0.71 nM versus
11, Ki = Ki = 0.109 nM. This trend indicates the secondary role of
the N-oxide group in binding as already reported.29,30
Since we try to identify the chemical features of antihyperalge-
sic activity of compounds with a pyrazolo[5,1-c][1,2,4]benzotri-
azine core 3, 8-disubstituted, the in vivo tests were conducted on
a sample of compounds representing all types of substitution.
Thus, the new compounds 4, 7, 11 and 15 were chosen to evaluate
the potential anxiolytic-like effects compared with those of diaze-
pam and antagonized by flumazenil (GABAA-R subtype antagonist).
Then, the antihyperalgesic effect in different pain models was
investigated. For these tests two already reported derivatives were
considered, in this paper numbered 933 and 10( )30 since they are
endowed with the 8-substituents suitable for our investigation:
the 8-pentyloxy group in compound 933 and the 8-(2-methyl-2-
phenylethoxy) group in 10( ).30
the pain threshold in the contralateral, non-operated paw, demon-
strating the lack of any analgesic effect.
Rats treated with 7, 9 and 15 (30 mg/kg po) in the same painful
condition did not exhibit any antihyperalgesic activity (data re-
ported in Supplementary data).
Compounds 4, 7, 9,33 10( ),30 11 and 15 were also tested in the
animal model of streptozotocin (STZ)-induced hyperalgesia which
reproduces the pain caused by diabetic neuropathy in laboratory
animals. STZ is particularly toxic for the insulin-producing beta
cells of the pancreas in mammals and it drastically increases the
serum glucose level. In diabetic patients hyperglycemia-induced
oxidative stress has been considered the main cause of diabetic
neuropathy.39Reactive oxygen species generated from an excess
shunting of glucose, leads to the formation and activation of pro-
tein kinase C and consequently to nervous tissue damage and pain.
Diabetic neuropathy can be due also to microvascular injury
involving small blood vessels that supply nerves in addition to
macrovascular conditions. It affects pain fibers, motor neurons
and the autonomic nerve. The identification of new molecules en-
dowed with activity against pain is very important since, despite
advances in the understanding of the metabolic causes of neurop-
athy, treatment to reduce pain is limited.
The hyperalgesia induced by STZ and its reversal caused by the
treatment with 4, 10( ),30 and 11 (30 mg/kg po) 15, 30, and 45 min
after their administration, is evidenced in the murine hot plate test
and after a single injection, Figure 3.
The antihyperalgesic effect remained up to 60 min for com-
pounds 10( ) and 11 (see complete data reported in the Supple-
mentary data). These molecules injected at the same doses
were not able to increase the pain threshold in the absence of
the STZ-induced painful condition. Derivatives 7, 9 and 15 were
devoid of any effect on the reduction of pain threshold provoked
by STZ.
Compounds 4 and 11 (10–30 mg/kg po), 7, 9, 10( )and 15 (30–
60 mg/kg po) were all unable to reduce the number of writhes in-
duced by ip injection of a solution of acetic acid, confirming the
lack of analgesic efficacy of these molecules, Table 3.
Protection from convulsions was evaluated in mice using penty-
lentetrazole (PTZ) as a chemical convulsant agent. All compounds
4, 7, 9,33 10( ),30 11 and (30 mg/kg po) and 15 (10 mg/kg po) were
devoid of any effect on PTZ-shock whereas diazepam (1 mg/kg ip),
the reference drug, completely protected against PTZ-induced
shocks and convulsions, Table 4.
The effects on mouse anxiety of newly synthesized molecules, 4
and 7 (3–30 mg/kg po), 11 (3–10 mg/kg po), 15 and 9,33 10( ),30
(10–30 mg/kg po), were studied using a light/dark box apparatus,
Figure 1a and b.
In our experiments, compounds 4 (10–30 mg/kg po), 7, 9,33
10( )30 and 15 (30 mg/kg po), 11 (10 mg/kg po), showed good anx-
iolytic-like effect with efficacy comparable to diazepam (standard
reference). The anxiolytic-like effects exhibited were completely
antagonized by flumazenil (at a dose of 100 mg/kg ip, a dose at
which flumazenil was able to antagonize the anxiolytic effect of
diazepam) suggesting that the anxiolytic-like effect of the newly
synthesized compounds is exerted through the GABAA subtype
receptors (through the benzodiazepine receptor).
Compounds 4, 7, 9,33 10( ),30 11 and 15 were tested on a pain
model in which peripheral mono-neuropathy was produced in
adult rats by loosely placing constrictive ligatures around the com-
mon sciatic nerve, according to the method described by Bennett
and Xie.37 The nociceptive threshold in the rat was determined
with an analgesimeter according to the paw pressure test.38 Fig-
ures 2a, b and c depict, for the sake of clarity, only graphics of
the active compounds (4, 10( ) and 11); results for all compounds
are reported in the Supplementary data.
Experiments were performed on rats submitted to the paw-
pressure test 14 days after the operation since at this time a signif-
icant reduction in the pain threshold of the injured paw (dx) was
observed. By contrast, in the contralateral paw the pain perception
remained unchanged. Compounds 4 (10–600 mg/kg po), 10( ) (10–
30 mg/kg po) and 11 (3–30 mg/kg po) showed statistical antihy-
peralgesic activity on this model when compared with the CMC
treated group, Figures 2a–c, respectively. The effect was observable
15, 30 and 45 min after administration for compounds 4 and 11
and up to 30 min for 10( ). Compound 11 exhibited a higher
potency since it exerted its protective effect starting from the
dose of 3 mg/kg po while the first active dose for the other
compounds, 4 and 10( ), was 10 mg/kg po (see Figs. 2a–c). The
above-mentioned compounds at the active doses did not modify
This result demonstrates that the mechanism of action respon-
sible for their antihyperalgesic effect is not identifiable with anti-
convulsant activity. This investigation was necessary because in
the scientific literature the ability of antiepileptic drugs to relieve
neuropathic painful conditions is widely reported.40–43
4. Conclusion
All the new compounds 3–8, 11–15 were designed to optimize a
previously published compound, III (chart 1) that showed the most
promising effects in the peripheral mono-neuropathy pain model
in adult rats. All new compounds were studied in vitro and com-
pounds 4, 7, 9,33 10( ),30 11 and 15 were chosen for in vivo tests.
In regard to structure–affinity relationships, the simultaneous
presence at position 8 of a –NH– moiety and at position 3 of iodine
atom, improve binding affinity, reaching a sub-nanomolar range,
compared to the 3-methyl derivatives. This indicates that it is very
important for receptor protein interaction the presence of a lipo-
philic group (iodine) and of a group that engages a hydrogen bond
(–NH–), as also evidenced in previous paper.30,36 In fact, the opti-
mization of the compound III30 was realized with the synthesis
of compound 11, 3-iodo-8-benzylaminopyrazolo[5,1-c][1,2,4] ben-
zotriazine 5-oxide, that stands out for its very high affinity value

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G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
Figure 1. (a and b) Anxiolytic like-effect of compounds 4, 7 and 9 (panel 1a) and 10( ), 11 and 15 (panel 1b) in comparison with diazepam (Daz, i.p.) on mouse light-dark box
test. The first column represents the number of transfers in 5 min, and the second column represents the time spent in light. Compound (p.o.) and diazepam were
administered 30 min and flumazenil (flu, i.p.) before the test. Each column represents the means SEM of 8 mice. ⁄P <0.01versus saline treated mice. §P <0.01 vs the
corresponding compound-treated mice. For sake of clarity compounds were divided in two panel.
(Ki = 0.109 nM) also with respect to its corresponding 3-methyl
derivative 7 (Ki = 2.58 nM).
antioxidants (including a-lipoic acid, dimethylthiourea, the xan-
thine oxidase inhibitor allopurinol and taurine), PKC- or PARP-
inhibitors, prevent thermal hyperalgesia in STZ-diabetic rodents
only after repeated administration.44 The efficacy of 4, 10( )
and 11 is comparable with that of lacosamide or kinin B1 recep-
tor antagonists that produced full reversal of thermal hyperalge-
sia.45 Moreover, 4, 10( ) and 11 show the antihyperalgesic
effect at a dosage comparable to that of other described sub-
type-selective benzodiazepine-site ligands endowed with anxio-
lytic-like effect.26
In the animal pain models (peripheral mono-neuropathy,
hyperalgesia STZ-induced) only compounds 4, 10( ),30 and 11
with selective anxiolytic profile, showed antihyperalgesic effects
at the dose of 10–30 mg/kg. Again compound 11, emerges be-
cause exhibits a higher potency since it exerted its protective ef-
fect at 3 mg/kg. It is important to note that compounds 4, 10( )
and 11 exerted their antihyperalgesic effect after a single injec-
tion while several substances, such as aldose reductase inhibitors,

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
7447
Figure 2. (a) Effect of 4 (1–60 mg/kg) in a rat model of mononeuropathy dx evaluated in the paw-pressure test. ⁄P <0.01, ^P <0.05.There were at least 8 rats per group. (b)
Effect of 10( ) (10–30 mg/kg) in a rat model of mononeuropathy dx evaluated in the paw-pressure test. ⁄P <0.01, ^P <0.05. There were at least four rats per group. (c) Effect of
11 (3-30 mg/kg) in a rat model of mononeuropathy dx evaluated in the paw-pressure test. ⁄P <0.01, ^P <0.05. There were at least four rats per group.

7448
G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
Figure 3. Streptozotocin (STZ) 200 mg/kg i.p. was dissolved in citrate buffer and injected 21 days before experiment. Each value represents the mean of at least 10
mice.⁄P<0.01 versus STZ-treated mice.
undesirable effects. This dual action on emotion end pain may be
Table 3
particularly useful therapeutically. Additional work is in progress
to gain more information on the receptor interaction (using our
pharmacophore ADLR procedure30) and to study in deep pharma-
cological aspects and the why some compounds with anxiolytic-
like effect (7, 9 and 15) have not antihyperalgesic effect.
Effect of compounds 4, 7, 9, 10, 11, 15 in the mouse abdominal constriction test
(acetic acid 0.6%)
Treatmenta
nb
Number of writhes
Compound
mg/kg p.o.
CMC 1%
4
15
9
32.4 2.5
31.2 3.5
27.3 3.2
29.1 2.4
27.4 2.6
30.7 2.7
28.5 2.8
31.6 3.1
27.3 2.6
26.3 3.1
26.1 2.5
28.8 2.9
27.5 3.0
10
30
30
60
30
60
30
60
10
30
30
60
5. Experimental section
5.1. Chemistry
10
10
10
10
10
8
9
10
8
7
9
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 chro-
matography, respectively. The structures of all compounds were
supported by their IR spectra (KBr pellets in nujol mulls, Perkin–
Elmer 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 Tables 1; all new compounds pos-
sess a purity P95%: microanalyses were performed with a Per-
kin–Elmer 260 analyzer for C, H, N. Mass spectra (m/z) were
recorded on a ESI-MS quadrupole (Varian 1200L) system, in posi-
tive ion mode, infusing a 10 mg/L solution of each analyte, dis-
solved in a mixture of mQ H2O:acetonitrile 1:1 v/v.
10
11
15
9
8
a
All drugs were administered 30 min before test.
Number of mice.
b
Table 4
Anticonvulsant activity of compounds 4, 7, 9, 10( ), 11, 15
Treatmenta
% Protection on PTZ-inducedb convulsions
Compound
mg/kg po
4
7
9
30
30
30
30
30
10
1
10
10
0
5.1.1. Sodium 2-cyanoprop-1-en-1-olate
A
solution of propionitrile (108 mmol) and ethylformate
10( )
11
15
0
(118 mmol) in anhydrous diethyl ether (20 mL) was added drop-
wise to a suspension of sodium hydride 50% (108 mmol) in anhy-
drous diethyl ether (250 mL) while temperature was maintained
below 30 °C. The reaction was stirred for 12–16 h and, after cool-
ing, the sodium salt was filtered and washed with anhydrous
diethyl ether solid. The sodium salt was immediately used for
10
10
100⁄
Diazepam
a
Treatment with new compounds and diazepam (ip) was performed 30 min
before the test.
b
Pentylenetetrazole (PTZ) 90 mg kgÀ1 ip Each value represents the mean of 10
mice.
the next reaction. IR
m
cmÀ1 2214. Anal C, H, N.
5.1.2. 1-(2-nitro-5-chlorophenyl)-2H-4-methyl-5-iminopyrazole
(1)
A suspension of sodium 2-cyanoprop-1-en-1-olate (7.0 mmol)
and ethanol (20 mL) was acidified with hydrochloride acid conc.
up to pH 2–3. The suspension was stirred and added of a solution
These findings represent a starting point for the development
of a novel class of ligands at GABAA subtype receptors with
antihyperalgesic effect since the synthesized compounds have
anxiolytic-like effect and have the potential to be therapeutic
agents for the relief of neuropathic pain, while being devoid of

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
7449
of 2-nitro-5-chlorophenylhydrazine46 (1.0 mmol) in ethanol
(20 mL); the reaction was stirred at refluxing temperature, for
2 h, and monitored by TLC until starting material disappearing.
The formed sodium chloride was filtered off and the ethanol solu-
tion was evaporated in vacuo. The crude dark red product was
washed by ether and characterized (870 mg, 35% yield). TLC elu-
8:2:1 v/v/v; 1H NMR (DMSO-d6) d 8.33 (d, J = 9.0 Hz, 1H, H-6); 8.13
(s, 1H, H-2); 7.74 (d, J = 2.8 Hz, 1H, H-9); 7.48 (d, J = 7.2 Hz, 1H, H-6’
Ph); 7.39 (t, J = 7.2 Hz, 1H, H-4’Ph); 7.30 (dd, J = 2.8, 9.0 Hz, 1H, H-7);
7.10 (d, J = 7.2 Hz, 1H, H-3’ Ph); 7.00 (t, J = 7.2 Hz, 1H, H-5’ Ph); 5.35
(s, 2H, CH2); 3.88 (s, 3H, OCH3); 2.26 (s, 3H, CH3). Anal C, H, N.
ent: toluene/ethyl acetate 8:2 v/v; IR
m
cmÀ13303; 1H NMR
5.1.8. 3-methyl-8-(2-methyl-2-phenylethoxy)pyrazolo[5,1-c]-
[1,2,4]benzotriazine 5-oxide (6( ))
(DMSO-d6) d 11.10 (s, 1H, @NH exchang.); 8.01 (br s, 1H, H-3);
7.80 (m, 2H, H-3’and H-6’); 6.90 (dd, J = 2.0, 9.0 Hz, 1H, H-4’);
4.10 (s, 1H, NH exchang.); 1.71 (s, 3H, CH3). Anal C, H, N.
From 3 and 2-phenylpropan-1-ol at 50 °C for 32 h; 34 mg, 34%
yield. Yellow crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; 1H NMR (DMSO-d6) dd 8.30 (d, J = 9.6 Hz, 1H, H-6);
8.12 (s, 1H, H-2); 7.61 (d, J = 2.8 Hz, 1H, H-9); 7.28–7.34 (m, 4H,
Ph); 7.21 (m, 2H, H-7 and Ph); 5.08 (m, 2H, CH2); 3.02 (m, 1H,
CH); 2.25 (s, 3H, 3-CH3); 1.35 (s, 3H, CH3). Anal C, H, N.
5.1.3. 1-(2-nitro-5-chlorophenyl)-4-methyl-5-aminopyrazole (2)
A suspension of sodium 2-cyanoprop-1-en-1-olate (7.0 mmol)
and ethanol (20 mL) was acidified with hydrochloride acid conc.
up to pH 2–3. The suspension was stirred and added of a solution
of 2-nitro-5-chlorophenylhydrazine46 (1.0 mmol) in ethanol
(20 mL); the reaction was stirred at refluxing temperature, for
10 h, and the imino derivative (1) was converted in the corre-
sponding 5-aminopyrazole 2. By TLC monitoring is possible to
evidence the spot of 5-aminopyrazole (2) at lower Rf than 5-
iminopyrazole (1); when the reaction was finished the ethanol
solution was evaporated and the final precipitate recrystallized
with ethanol (1.05 g, 42% yield). Yellow crystals; TLC eluent: tolu-
5.1.9. General procedure for the synthesis of 7–8, 11–15
A solution of the starting material 3 (0.35 mmol) or 3-iodo-8-chlo-
ropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide,34 in ethanol (5.0 mL)
was added of an excess of suitable amine (1.0 mL) and refluxed under
vigorous stirring (time specified for each compound). The reaction was
monitored by TLC, and when the starting material disappeared, the fi-
nal precipitate was filtered, or in case of a solution, ice was added to ob-
tain a precipitate; then the crude residue was washed with isopropyl
ether and recrystallized by suitable solvent.
ene/ethyl acetate 8:2 v/v; IR
m
cmÀ1 3299; 1H NMR (DMSO-d6) d
8.10 (d, J = 9.1 Hz, 1H, H-3’); 7.85 (d, J = 1.9 Hz, 1H, H-6’); 7.76
(dd, J = 1.9, 9.1 Hz, 1H, H-4’); 7.38 (s, 1H, H-3); 3.80 (br s, 2H,
NH2 exchang.); 1.85 (s, 3H, CH3). Anal C, H, N.
5.1.10. 3-methyl-8-benzylaminopyrazolo[5,1-
c][1,2,4]benzotriazine 5-oxide (7)
From 3 and benzyl amine for 48 h; 42.7 mg, 40% yield. Yellow
crystals; TLC and chromatography column eluent: toluene/ethyl
acetate 8:2 v/v; IR
1H, NH, exchang.); 8.11 (d, J = 8.9 Hz, 1H, H-6); 8.01 (s, 1H, H-2);
7.42–7.38 (m, 4H, Ph); 7.28 (m, 1H, Ph); 7.04 (dd, J = 2.7, 8.9 Hz,
1H, H-7); 6.80 (d, 2.7 Hz, 1H, H-9); 4.55 (d, J = 5.2 Hz, 2H, CH2);
2.26 (s, 3H, CH3). Anal C, H, N.
5.1.4. 3-methyl-8-cloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 3
The 5-iminopyrazole (1) or the 5-aminopyrazole (2) (1.0 mmol)
dissolved in 1 mL of diglyme (diethylene glycol dimethyl ether)
and 30 mL of 10% sodium hydroxide solution was added. The reac-
tion was maintained at 50 °C under stirring for 12 h. The final sus-
pension was filtered and the crude yellow product was
recrystallized by suitable solvent (176 mg, 75% yield). Yellow crys-
m
cmÀ1 3271; 1H NMR (DMSO-d6) d 8.20 (m,
tals; TLC eluent: isopropyl ether/ciclohexane 8:3 v/v; IR
m
cmÀ1
5.1.11. 3-methyl-8-(2-methoxybenzylamino)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (8)
1580; 1H NMR (DMSO-d6) d 8.41 (d, J = 9.2 Hz, 1H, H-6); 8.31 (d,
J = 2.4 Hz, 1H, H-9); 8.17 (s, 1H, H-2); 7.72 (dd, J = 2.4, 9.2 Hz, 1H,
H-7); 2.35 (s, 3H, CH3). Anal C, H, N.
From 3 and 2-methoxybenzylamine for 56 h; 64.5 mg, 55%
yield. Dark yellow crystals; TLC and chromatography column elu-
ent: toluene/ethyl acetate 8:2 v/v; IR
m
cmÀ1 3271; 1H NMR
5.1.5. General procedure for the synthesis of 4–6( )
(DMSO-d6) d 8.11 (d, J = 9.0 Hz, 1H, H-6); 8.03 (m, 1H, NH, ex-
chang.); 8.00 (s, 1H, H-2); 7.27 (m, 2H, H-4 and H-6 Ph); 7.09 (m,
2H, H-9 and H-3 Ph); 6.97 (dd, J = 2.8, 9.0 Hz, 1H, H-7); 6.92 (t,
J = 6.9 Hz, 1H, H-5 Ph); 4.45 (d, J = 5.2 Hz, 2H, CH2); 3.90 (s, 3H,
OCH3); 2.20 (s, 3H, CH3). Anal C, H, N.
The reaction was carried out in 10 mL of dichloromethane to
which was added the starting material 3 (0.3 mmol), 5 mL of 40%
sodium hydroxide solution, 0.5 mmol of tetrabutylammonium bro-
mide, and the suitable alcohol in large excess (ratio with starting
material 1:15) under vigorous stirring (temperature and time spec-
ified for each compound). The reaction was monitored by TLC, and
when the starting material disappeared, the organic layer was sep-
arated and the aqueous layer extracted twice with 10 mL of dichlo-
romethane. The combined organic extracts were evaporated and
the residue was recovered with isopropil ether and then recrystal-
lized by suitable solvent.
5.1.12. 3-iodo-8-benzylaminopyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (11)
From 3-iodo-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide,34 and benzyl amine for 36 h; 119.7 mg, 82% yield. Yellow
crystals; TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1 v/v/v;
IR
m
cmÀ13270; 1H NMR (DMSO-d6)) d 8.32 (m, 1H, NH, exchang.);
8.22 (s, 1H, H-2); 8.12 (d, J = 8.9 Hz, 1H, H-6); 7.40–7.36 (m, 4H,
Ph); 7.27 (m, 1H, Ph); 7.02 (m, 2H, H-7 and H-9); 4.52 (d,
J = 5.2 Hz, 2H, CH2). Anal C, H, N.
5.1.6. 3-methyl-8-benzyloxypyrazolo[5,1-c][1,2,4]benzotriazine
5-oxide (4)
From 3 and benzyl alcohol, at 50 °C for 32 h; 52 mg, 57% yield.
Yellow crystals; TLC eluent: ciclohexane/ethyl acetate 2:1 v/v; 1H
NMR (DMSO-d6) d 8.35 (d, J = 9.6 Hz, 1H, H-6); 8.13 (s, 1H, H-2);
7.74 (d, J = 2.1 Hz, 1H, H-9); 7.53 (d, J = 7.6 Hz, 2H, Ph); 7.42 (t,
J = 7.6 Hz, 2H, Ph); 7.39 (t, J = 7.6 Hz, 1H, Ph); 7.32 (dd, J = 2.1,
9.6 Hz, 1H, H-7); 5.42 (s, 2H, CH2); 2.26 (s, 3H, CH3). Anal C, H, N.
5.1.13. 3-iodo-8-(2-methoxybenzylamino)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (12)
From 3-iodo-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide,34 and 2-methoxybenzylamine for 16 hours; 99.8 mg, 64%
yield. Orange crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; IR
m
cmÀ13302; 1H NMR (DMSO-d6) d 8.22 (s, 1H, H-2);
5.1.7. 3-methyl-8-(2-methoxybenzyloxy)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (5)
From 3 and 2-methoxybenzyl alcohol, at 40 °C for 24 h; 52 mg, 52%
yield. Yellow crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8.18 (m, 1H, NH, exchang.); 8.11 (d, J = 9.1 Hz, 1H, H-6); 7.29 (m,
2H, H-4 and H-6 Ph); 7.09 (m, 2H, H-9 and H-3 Ph); 7.03 (dd,
J = 2.6, 9.1 Hz, 1H, H-7); 6.92 (t, J = 7.0 Hz, 1H, H-5 Ph); 4.45 (d,
J = 5.2 Hz, 2H, CH2); 3.90 (s, 3H, OCH3). Anal C, H, N.

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G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
5.1.14. 3-iodo-8-(4-chlorobenzylamino)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (13)
5.1.20. 3-iodo-8-benzylaminopyrazolo[5,1-c][1,2,4]-
benzotriazine (11R)
From 3-iodo-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide,34 in DMF (2.0 mL) and 4-chlorobenzylamine for 48 hours;
94.8 mg, 60% yield. Orange crystals; TLC eluent: toluene/ethyl ace-
From 11; 66.2 mg, 55% yield. Yellow crystals; TLC eluent: ethyl
acetate/petroleum benzine 2:1 v/v; IR
(DMSO-d6) d 8.45 (m, 1H, NH, exchang.); 8.35 (s, 1H, H-2); 8.25
(d, J = 8.9 Hz, 1H, H-6); 7.48–7.36 (m, 4H, Ph); 7.33 (m, 1H, Ph);
7.28 (d, J = 2.3 Hz, 1H, H-9); 7.22 (dd, J = 2.3, 8.9 Hz, 1H, H-7);
4.60 (d, 2H, CH2); ESI-MS: found m/z 402.1 [M+H]+. Anal C, H, N.
m
cm1 3337; 1H NMR
tate/acetic acid 8:2:1 v/v/v; IR
m
cmÀ1 3304; 1H NMR (DMSO-d6)) d
8.32 (m, 1H, NH, exchang.); 8.27 (s, 1H, H-2); 8.12 (d, J = 9.0 Hz, 1H,
H-6); 7.43 (m, 4H, Ph); 7.02 (m, 2H, H-7 and H-9); 4.55 (d,
J = J = 5.2 Hz, 2H, CH2). Anal C, H, N.
5.2. Radioligand binding assay
5.1.15. 3-iodo-8-(2-phenylethylamino)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (14)
[3H]Ro15–1788 (specific activity 78.8 Ci/mmol) was obtained
from Perkin Elmer. All the other chemicals, which were of reagent
grade, were obtained from commercial suppliers.
From 3-iodo-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide,34 and phenylethylamine for 10 hours; 120.7 mg, 80% yield.
Yellow crystals; TLC eluent: toluene/ethyl acetate/acetic acid
Bovine cerebral cortex membranes were prepared as previously
described.47,48 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.49 [3H]Ro 15–1788 binding studies were performed as previ-
ously reported.50 At least six different concentrations of each com-
pound were used. The data of n = 5 experiments carried out in
triplicate were analyzed by means of an iterative curve-fitting pro-
cedure (program Prism, GraphPad, San Diego, CA), which provided
IC50, Ki, and SEM values for tested compounds, the Ki values being
calculated from the Cheng and Prusoff equation.51
8:2:1 v/v/v; IR
m
cmÀ1 3303; 1H NMR (DMSO-d6) d 8.27 (s, 1H, H-
2); 8.12 (d, J = 8.9 Hz, 1H, H-6); 7.88 (m, 1H, NH, exchang.); 7.33
(m, 4H, Ph); 7.24 (m, 1H, Ph); 7.08 (d, J = 2.7 Hz, 1H, H-9); 6.98
(dd J = 2.7, 8.9 Hz, 1H, H-7); 3.53 (t, J = 5.0 Hz, 2H, CH2NH); 2.96
(t, J = 5.0 Hz, 2H, CH2Ph). Anal C, H, N.
5.1.16. 3-iodo-8-(benzyl(methyl)amino)pyrazolo[5,1-c][1,2,4]-
benzotriazine 5-oxide (15)
From 3-iodo-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-
oxide,34 and N-methylbenzylamine for 10 hours; 84.5 mg, 56%
yield. Yellow crystals; TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v; IR
m
cmÀ1 3303; 1H NMR1H NMR (DMSO-d6) d 8.25 (s,
5.3. Pharmacological methods
1H, H-2); 8.18 (d, J = 9.1 Hz, 1H, H-6); 7.37 (m, 2H, H-2 and H-6
Ph); 7.26 (m, 3H, H-3, H-4 and H-5 Ph); 7.16 (m, 2H, H-6 and H-
9); 4.90 (s, 2H, CH2); 3.33 (s, 3H, N-CH3). Anal C, H, N.
Animals: Male Swiss albino mice (23–30 g) and male Sprague–
Dawley rats from Harlan- (Varese) breeding farm were used. Fif-
teen mice or four rats were housed per cage. The cages were placed
in the experimental room 24 h before the test for acclimatisation.
The animals were kept at 23 1 °C with a 12 h light/dark cycle,
light at 7 a.m., with food and water ad libitum. All experiments
were carried out according to the guidelines of the European Com-
munity Council.
5.1.17. General procedure for the synthesis of 4R, 7R and 11R
A solution of suitable 5-oxide derivative 4, 7 and 11 (0.3 mmol)
in acetic acid (10 mL) was magnetically stirred at room tempera-
ture. A large excess of zinc dust (2.0 mmol in three portions) and
2 Â 5 mL of acetic acid were added. After 15 min the reaction
was stopped, since the starting material disappeared (evaluated
by TLC, eluent ethyl acetate/ petroleum benzine 2:1 v/v) and the
zinc residue was filtered off. The solution was evaporated in vacuo
and the final sugary residue was treated with ice/water. In case of
derivative 4R the aqueous solution was extracted with ethyl ace-
tate and, after anhydrification with sodium sulfate anhydrous,
evaporated. The residue was recovered with di-isopropyl ether, fil-
tered and recrystallized by suitable solvent. In case of derivatives
7R and 11R the aqueous solution was made slightly alkaline (pH
7–8) to permit the final compounds to precipitate. Also in this case,
the precipitate was filtered and recrystallized by suitable solvent.
5.3.1. Pentylenetetrazole (PTZ)-induced seizure
PTZ (90 mg/kg s.c.) was injected 30 min after the administration
of drugs. The frequency of the occurrence of clonic generalized
convulsions was noted over a period of 30 min.
5.3.2. 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 Â 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.
5.1.18. 3-methyl-8-benzyloxypyrazolo[5,1-c][1,2,4]-
benzotriazine (4R)
From 4; 59.2 mg, 68% yield. Yellow crystals; TLC eluent: ethyl
acetate/petroleum benzine 2:1 v/v; 1H NMR (CDCl3) d 8.50 (d,
J = 9.2 Hz, 1H, H-6); 8.05 (s, 1H, H-2); 8.38 (d, J = 2.4 Hz, 1H, H-9);
7.52 (d, J = J = 7.2 Hz, 2H, Ph); 7.44 (t, J = 7.2 Hz, 2H, Ph); 7.39 (t,
J = 7.2 Hz, 1H, Ph); 7.33 (dd, J = 2.4, 9.2 Hz, 1H, H-7); 5.32 (s, 2H,
CH2); 2.69 (s, 3H, CH3); ESI-MS: found m/z 291.1 [M+H]+. Anal C, H, N.
5.3.3. Hot plate test
The method adopted was described by O’Callaghan and Holz-
man.52 Mice were placed inside a stainless steel container, thermo-
statically set at 52.5 0.1 °C in a precision water-bath from KW
Mechanical Workshop, Siena, Italy. Reaction times (s), were mea-
sured with a stop-watch before i.c.v. injections and at regular inter-
vals (15 min) up to a maximum of 60 min after treatment (cut-off)
in order to prevent tissue damage. The endpoint used was the lick-
ing of the fore or hind paws. Antinociception was seen as increased
latencies to the responses evaluated, while increased nociception
was seen by shorter latencies. Those mice scoring below 12 and
5.1.19. 3-methyl-8-benzylaminopyrazolo[5,1-c][1,2,4]-
benzotriazine (7R)
From 7; 62.5 mg, 72% yield. Pale orange crystals; TLC eluent:
ethyl acetate/petroleum benzine 2:1 v/v; IR
m
cm1 3270; 1H NMR
(CDCl3) d 8.29 (d, J = 8.8 Hz, 1H, H-6); 7.89 (s, 1H, H-2); 7.41 (m,
4H, Ph); 7.37 (m, 1H, Ph); 7.32 (d, J = 2.4 Hz, 1H, H-9); 6.93 (dd,
J = 2.4, 8.8 Hz, 1H, H-7); 5.11 (m, 1H, NH, exchang.); 4.57 (d,
J = 5.2 Hz, 2H, CH2); 2.65 (s, 3H, CH3); ESI-MS: found m/z 290.2
[M+H]+. Anal C, H, N.

G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
7451
over 18 s in the pretest were rejected (30%). An arbitrary cut-off
5.5. Statistical analysis
time of 45 s was adopted.
All experimental results are given as the mean S.E.M. Analysis
of variance (ANOVA), followed by Fisher’s Protected Least Signifi-
cant Difference (PLSD) procedure for post-hoc comparison, was
used to verify significance between two means. Data were ana-
lysed with the StatView software for the Macintosh (1992). P val-
ues of less than 0.05 were considered significant.
5.3.4. Abdominal constriction test
Mice were injected i.p. with a 0.6% solution of acetic acid
(10 ml kgÀ1), according to Koster.53 The number of stretching
movements was counted for 10 min, starting 5 min after acetic
acid injection.
Supplementary data
5.3.5. Paw pressure test
The nociceptive threshold in the rat was determined with an
analgesimeter (Ugo Basile, Varese, Italy), according to the method
described by Leighton.38The instrument exerts a force which is ap-
plied at a costant rate (32 g per second) with a cone-shaped pusher
on the upper surface of the rat hind paw. The force is continuously
monitored by a pointer moving along a linear scale. The pain
threshold is given by the force which induces the first struggling
from the rat. Pretested rats which scored below 40 g or over 75 g
during the test before drug administration (25%) were rejected.
An arbitrary cut off value of 250 g was adopted.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.10.047.
References and notes
1. Fritschy, J.-M.; Crestani, F.; Rudolph, U.; Mohler, H. GABAA receptor subtypes
Memory function and neurological disorders. In Excitatory-Inibitory Balance
synapses, circuit, systems; Hensch, T. K., Fagiolini, M., Eds.; Kluwer Academic/
Plenum: New York, 233 Spring Street, New York 10013, 2004; pp 215–228.
2. Olsen, R. W.; Sieghart, W. Neuropharmacology 2009, 56, 141–148.
3. Collingridge, G. L.; Olsen, R. W.; Peters, J.; Spedding, M. Neuropharmacology
2009, 56, 2–5.
4. Sieghart, W. Adv. Pharmacol. 2006, 54, 231–263.
5.3.6. Chronic Constriction Injury (CCI)
5. Enna, S. J.; McCarson, K. E. Adv. Pharmacol. 2006, 54, 1–27.
6. Zeilhofer, H. U. Int. Immunopharmacol. 2008, 8, 182–187.
7. Price, T. J.; Cervero, F.; Gold, M. S.; Hammond, D. L.; Prescott, S. A. Brain Res. Rev.
2009, 60, 149–170.
8. Munro, G.; Ahring, P. K.; Mirza, N. R. TiPS 2009, 30, 453–459.
9. Hammond, D. L. Regional Anesthesia and Pain Medicine 2001, 26, 551–557.
10. Zeilhofer, H. U.; Zeilhofer, U. B. Neurosci. Lett. 2008, 437, 170–174.
11. Jourdan, D.; Ardid, D.; Bardin, L.; Bardin, M.; Neuzeret, D.; Lanphouthacoul, L.;
Eschalier, A. Pain 1997, 71, 265–270.
12. Luger, T. J.; Hayashi, T.; Weiss, C. G.; Hill, H. F. Eur. J.Pharmacol. 1995, 275, 153–
162.
13. Kohno, T.; Wakai, A.; Ataka, T.; Ikoma, M.; Yamakura, T.; Baba, H. ‘Actions of
Midazolam on Excitatory Transmission in Dorsal Horn Neurons of Adult Rat
Spinal Cord’; Anesthesiology, 2006.
A peripheral mono neuropathy was produced in adult rats by
placing loosely constrictive ligatures around the common sciatic
nerve according to the method described by Bennett.37 Rats were
anaesthetised with chloral hydrate. The common sciatic nerve
was exposed at the level of the middle of the thigh by blunt dissec-
tion through biceps femoris. Proximal to sciatica’s tri furcation,
about 1 cm of the nerve was freed of adhering tissue and four liga-
tures (3/0 silk tread) were tied loosely around it with about 1 mm
spacing. The length of the nerve thus affected was 4–5 mm long.
Great care was taken to tie the ligatures such that the diameter
of the nerve was seen to be just barely constricted when viewed
with 40x magnification. In every animal, an identical dissection
was performed on the opposite side except that the sciatic nerve
was not ligated. The left paw was untouched.
14. Batra, Y. K.; Mahajan, R.; Kumar, S.; Rajeev, S.; Singh Dhillon, M. Anesth. Analg.
2008, 107, 669–672.
15. Huffman, J. C.; Stern, T. A. J. Emerg. Med. 2003, 25, 427–437.
16. Nishiyama, T. Can. J. Anesthesia 2006, 53, 1004–1009.
17. Rudolph, U.; Crestani, F.; Benke, D.; Brünig, I.; Benson, J. A.; Fritschy, J. M.;
Martin, J. R.; Bluethmann, H.; Möhler, H. Nature 1999, 401, 796–800.
18. McKernan, R. M.; Rosahl, T. W.; Reynolds, D. S.; Sur, C.; Wafford, K. A.; Atack, J. R.;
Farrar, S.; Myers, J.; Cook, G.; Ferris, P.; Garrett, L.; Bristow, L.; Marshall, G.; Macaulay,
A.; Brown, N.; Howell, O.; Moore, K. W.; Carling, R. W.; Street, L. J.; Castro, J. L.;
Ragan, C. I.; Dawson, G. R.; Whiting, P. J. Nat. Neurosci. 2000, 3, 587–592.
19. Reynolds, D. S. Pharmacol., Biochem. Behav. 2008, 90, 37–42.
20. Löw, K.; Crestani, F.; Keist, R.; Benke, D.; Brünig, I.; Benson, J. A.; Fritschy, J. M.;
Rülicke, T.; Bluethmann, H.; Möhler, H.; Rudolph, U. Science 2000, 290, 131–
134.
21. Dias, R.; Sheppard, W. F. A.; Fradley, R. L.; Garrett, E. M.; Stanley, J. L.; Tye, S. J.;
Goodacre, S.; Lincoln, R. J.; Cook, S. M.; Conley, R.; Hallett, D.; Humphries, A. C.;
Thompson, S. A.; Wafford, K. A.; Street, L. J.; Castro, J. L.; Whiting, P. J.; Rosahl, T.
W.; Atack, J. R.; McKernan, R. M.; Dawson, G. R.; Reynolds, D. S. J. Neurosci.
2005, 25, 10682–10688.
22. Maubach, K. Curr Drug Targets CNS Neurol Disord 2003, 2, 233–239.
23. Atack, J. R. Curr. Top. Med. Chem. 2011, 11, 1176–1202.
24. Knabl, J.; Witschi, R.; Hosl, K.; Reinold, H.; Zeilhofer, U. B.; Ahmadi, S.;
Brockhaus, J.; Sergejeva, M.; Hess, A.; Brune, K.; Fritschy, J.-M.; Rudolph, U.;
Mohler, H.; Zeilhofer, H. U. Nature 2008, 451, 330–334.
25. Knabl, J.; Zeilhofer, U. B.; Crestani, F.; Rudolph, U.; Zeilhofer, H. U. Pain 2009,
141, 233–238.
26. Zeilhofer, H. U.; Möhler, H.; Di Lio, A. TiPS 2009, 30, 397–402.
27. Mirza, N. R.; Larsen, J. S.; Mathiasen, C.; Jacobsen, T. A.; Munro, G.; Erichsen, H.
K.; Nielsen, A. N.; Troelsen, K. B.; Nielsen, E. O.; Ahring, P. K. J. Pharmacol. Exp.
Ther. 2008, 327, 954–968.
5.3.7. Induction of diabetes (streptozotocin Treatment)
Mice were injected intravenously in the tail with 200 mg kgÀ1
of streptozotocin (STZ) prepared in saline adjusted to pH 4.5 in
0.1 N citrate buffer. Non-diabetic control mice were injected with
the vehicle alone. STZ solutions were freshly prepared due to the
limited stability of the compound. Rashid & Ueda,54 in a set of pre-
liminary control experiments, measured spectro- photometrically
serum glucose level at 7, 14, and 21 days after streptozotocin treat-
ment. They found that it was consistent with a diabetic level
(above 300 mg/dl) throughout the periods. The serum glucose level
was measured by glucose oxidase method from blood samples. The
animals were found to develop significant thermal hyperalgesia at
the 3rd week after streptozotocin treatment. In the pre-test per-
formed in the test day those mice scoring over 20 s in hot plate test
were rejected. Test was performed 21 days post streptozotocin
treatment at 30, 60, 90 and 120 min after the last injection of test
compounds.
5.4. Drugs
28. Di Lio, A.; Benke, D.; Besson, M.; Desmeules, J.; Daali, Y.; Wang, Z.-J.; Edwankar,
R.; Cook, J. M.; Zeilhofer, H. U. Neuropharmacology 2011, 60, 626–632.
29. Costanzo, A.; Guerrini, G.; Ciciani, G.; Bruni, F.; Costagli, C.; Selleri, S.; Besnard,
F.; Costa, B.; Martini, C.; Malmberg-Aiello, P. J. Med. Chem. 2002, 45, 5710–
5720.
30. Guerrini, G.; Ciciani, G.; Cambi, G.; Bruni, F.; Selleri, S.; Guarino, C.; Melani, F.;
Montali, M.; Martini, C.; Ghelardini, C.; Norcini, M.; Costanzo, A. J. Med. Chem.
2009, 52, 4668–4682.
31. Guerrini, G.; Ciciani, G.; Bruni, F.; Selleri, S.; Guarino, C.; Melani, F.; Montali, M.;
Daniele, S.; Martini, C.; Ghelardini, C.; Norcini, M.; Ciattini, S.; Costanzo, A. J.
Med. Chem. 2010, 53, 7532–7548.
32. Guerrini, G.; Ciciani, G.; Cambi, G.; Bruni, F.; Selleri, S.; Melani, F.; Montali, M.;
Martini, C.; Ghelardini, C.; Norcini, M.; Costanzo, A. Bioorg. Med. Chem. 2008, 16,
4471–4489.
Diazepam (Valium 10–Roche), flumazenil (Roche), pentylen-
etetrazole (PTZ) (Sigma), zolpidem (Tocris) were used. All drugs ex-
cept PTZ were suspended in 1% carboxymethylcellulose sodium
salt and sonicated immediately before use. PTZ was dissolved in
isotonic (NaCl 0.9%) saline solution and injected s.c. All benzodiaz-
epine receptor ligands were administered by po route, except for
flumazenil which was administered i.p. Drug concentrations were
prepared in such a way that the necessary dose could be adminis-
tered in a volume of 10 ml/kg by the po, i.p. or s.c. routes.

7452
G. Guerrini et al. / Bioorg. Med. Chem. 19 (2011) 7441–7452
33. Guerrini, G.; Ciciani, G.; Cambi, G.; Bruni, F.; Selleri, S.; Besnard, F.; Montali, M.;
Martini, C.; Ghelardini, C.; Galeotti, N.; Costanzo, A. Bioorg. Med. Chem. 2007,
15, 2573–2586.
43. Colombo, E.; Francisconi, S.; Faravelli, L.; Izzo, E.; Pevarello, P. Future Med.
Chem. 2010, 2, 803–842.
44. Obrosova, I. G. Neurotherapeutics 2009, 6, 638–647.
34. Costanzo, A.; Guerrini, G.; Ciciani, G.; Bruni, F.; Costagli, C.; Selleri, S.; Costa, B.;
Martini, C.; Malmberg-Aiello, P. Med. Chem. Res. 2002, 11, 87–101.
35. Hansch, C.; Leo, A.; Hoeckman, D. Exploring Qsar, Hydrophobic, Electronic And
Steric Constant; ACS Professional References Book, American Chemical Society:
Washington, D. C., 1995.
45. Beyreuther, B.; Callizot, N.; Stohr, T. Eur. J. Pharmacol. 2006, 539, 64–70.
46. Lindley, J. M.; McRobbie, I. M.; Meth-Chon, O.; Suschitzky, H. J. Chem. Soc. Perkin
Trans 1 1980, 982–994.
47. Martini, C.; Lucacchini, A.; Ronca, G.; Hrelia, S.; Rossi, C. A. J. Neurochem. 1982,
38, 15–19.
36. Guerrini, G.; Ciciani, G.; Bruni, F.; Selleri, S.; Melani, F.; Daniele, S.; Martini, C.;
Costanzo, A. Bioorg. Med. Chem. 2011, 19, 3074–3085.
37. Bennett, G. J.; Xie, Y. K. Pain 1988, 33.
48. Primofiore, G.; Da Settimo, F.; Taliani, S.; Marini, A. M.; Novellino, E.; Greco, G.;
Lavecchia, A.; Besnard, F.; Trincavelli, L.; Costa, B.; Martini, C. J. Med. Chem.
2001, 44, 2286–2297.
38. Leighton, G. E.; Rodriguez, R. E.; Hill, R. G.; Huges, J. Br. J.Pharmacol 1988, 93,
533–560.
39. Kitada, M.; Koya, D.; Sugimoto, T.; Isono, M.; Araki, S.-I.; Kashiwagi, A.; Haneda,
M. Diabetes 2003, 52.
40. Dworkin, R. H.; Backonja, M.; Rowbotham, M. C.; Allen, R. R.; Argoff, C. R.;
Bennett, G. J.; Bushnell, M. C.; Farrar, J. T.; Galer, S. B.; Haythornthwaite, J. A.;
Hewitt, D. J.; Loeser, J. D.; Max, M. B.; Saltarelli, M.; Schmader, K. E.; Stein, C.;
Thompson, D.; Turk, D. C.; Wallace, M. S.; Watkins, L. R.; Weinstein, S. M. Arch.
Neurol. 2003, 60, 1524–1534.
49. Lowry, O. H.; Rosenbrough, N. J.; Farr, A.; Barnard, E. A.; Skolnick, P.; Olsen, R.
W.; Möhler, H.; Sieghart, W.; Biggio, G.; Braestrup, C.; Bateson, A. N.; Langer, S.
Z. L.; Randali, R. J. J. Biol. Chem 1951, 193, 265–275.
50. Costanzo, A.; Guerrini, G.; Ciciani, G.; Bruni, F.; Selleri, S.; Costa, B.; Martini, C.;
Lucacchini, A.; Malmberg-Aiello, P.; Ipponi, A. J. Med. Chem 1999, 42, 2218–
2226.
51. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol 1973, 22, 3099–3108.
52. O’Callaghan, J. P.; Holtzman, S. G. J. Pharmacol. Exp. Ther. 1975, 192, 497–505.
53. Koster, R.; Anderson, M.; De Beer, E. J. Fed. Proc. 1959, 18, 412.
54. Rashid, M. H.; Ueda, H. J. Pharmacol. Exp. Ther. 2002, 303, 226–231.
41. Owen, R. T. Drugs Today 2007, 43, 857–863.
42. Dib-Hajj, S. D.; Black, J. A.; Waxman, S. G. Pain Med. 2009, 10, 1260–1269.