Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as high affinity GABAA-R ligands and potential anxiolytics

Article abstract of DOI:10.1016/j.bmc.2018.06.021

The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM) of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs started from anilines and diethyl 2-(ethoxymethylene)malonate to give the q

Full text of DOI:10.1016/j.bmc.2018.06.021

Accepted Manuscript
Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as
high affinity GABAA-R ligands and potential anxiolytics
Marisa J. López Rivilli, Anahí V. Turina, Elena A. Bignante, Victor H. Molina,
María A. Perillo, Margarita C. Briñon, Elizabeth L. Moyano
PII:
S0968-0896(18)30231-1
https://doi.org/10.1016/j.bmc.2018.06.021
BMC 14413
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
4 February 2018
20 May 2018
14 June 2018
Please cite this article as: López Rivilli, M.J., Turina, A.V., Bignante, E.A., Molina, V.H., Perillo, M.A., Briñon,
M.C., Moyano, E.L., Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as high affinity
GABAA-R ligands and potential anxiolytics, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.06.021
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Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as
high affinity GABAA-R ligands and potential anxiolytics
Marisa J. López Rivillia, Anahí V. Turinab, Elena A. Bignantec, Victor H. Molinac, María A.
Perillob, Margarita C. Briñond*, Elizabeth L. Moyanoa*
aUniversidad Nacional de Córdoba, Facultad de Ciencias Químicas Departamento de Química
Orgánica and CONICET, Instituto de Investigaciones en Fisico-Química de Córdoba (INFIQC),
Córdoba, X5000HUA, Argentina.
bUniversidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales,
Departamento de Química, Biofísica-Química, Cátedra de Química Biológica and CONICET,
Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT), Córdoba, 5000, Argentina.
cUniversidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de
Farmacología, Córdoba, X5000HUA, Argentina.
dUniversidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de Ciencias
Farmacéuticas, Córdoba, X5000HUA, Argentina.
Corresponding author E-mail address: lauramoy@fcq.unc.edu.ar
Dedicated to our beloved Prof. Gloria Inés Yranzo on the tenth anniversary of her physical
departure.
Abstract: The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM)
of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs
started from anilines and diethyl 2-(ethoxymethylene)malonate to give the quinolin-4-one
1

nucleus, via the Gould-Jacobs reaction. These quinolinones were transformed to 4-
chloroquinolines, which react with aryl-hydrazines affording the final compounds. PQs exhibited
different potency in displacing specific [H3]Flunitrazepam binding from the benzodiazepine
binding site at the -aminobutiric acid receptor (GABAA-R) depending on the substitution of the
pyrazoloquinolone nucleus. PCA helped determine how different substituents contributed to the
differential behavior of the PQs studied. Compounds with high affinity for the GABAA-R were
tested regarding their anxiolytic properties in Wistar adult male rats using the Elevated Plus Maze
(EPM). Thus, PQs with a p-methoxy phenyl group at N-1 (7b-ii and 7c-ii) displayed a
remarkable anxiolytic activity at low doses (0.5-1.0 mg/kg). Meanwhile, PQs featuring an
unsubstituted phenyl (7b-i) or p-fluoro phenyl group (7b-iii) at the N-1 showed anxiogenic
effects in the EPM test.
Keywords: pyrazoloquinolinones; elevated plus maze; GABAA receptor; benzodiazepine binding
1. Introduction
Among the pyrazole derivatives, the condensed compounds having the pyrazoloquinolin-
3-one (PQ) core have extensively been studied for their biological activities e.g.
acetylcholinesterasa inhibitors, anti-inflammatory agents, antimalarial and antiviral activities,
between others [1-3].
In particular, PQs have been known for their high affinity for the benzodiazepine binding
site (BZDBS) at the ionotropic -aminobutiric acid (GABA) receptor (GABAA-R) [4-6]. After the
performance of PQs was described, there has been a growing interest in their peculiar
pharmacological activity. These target compounds constitute a biologically interesting class of
2

ligands, lacking some unwanted side effects (e.g. sedative effects) found in classical
benzodiazepines [7], as well as the net shift of intrinsic activity from agonist, through antagonist,
to inverse agonist, caused by small structural changes render the PQs a fascinating and intriguing
class of GABAA-R ligands [5,8-9]. GABAA-R belongs to the “Cys-loop ligand-gated ion
channels” family, specifically a chloride channel gated by the -aminobutiric acid, the most
widely distributed inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates
[10,11]. Thus, GABAA-R is the target of many pharmacologically relevant drugs and possess
several allosteric binding sites which modulate in diverse ways the effect of its endogenous agonist
GABA [12]. These allosteric modulators recognize own specific binding sites in the receptor,
different from the specific GABA site [13]. Thus, the chloride flux is increased by several types of
depresant drugs, fundamentally benzodiazepines (BZDs), barbiturates, steroids and anaesthetics,
whereas it is reduced by convulsant agents like bicuculline and picrotoxinin [14,15].
The most important drugs in clinical use for GABAA-R are the BZDs, which mainly have
anxiolytic, anticonvulsivant, muscle relaxant and sedative-hypnotic-effects [11,15]. For this
reason, the BZDs and other chemical compounds capable of bindings with high affinity to
GABAA-R are considered therapeutic agents of the upmost importance to treat specific diseases
in the CNS [16]. Since the discovery of chlordiazepoxide and diazepam [17] the 1,4-
benzodiazepines, have been fruitful source of research however, anxioselective compounds,
which do not possess the undesirable side effects of BZDs are needed [18,19].
In this regard, 2-aryl-pyrazolo[4,3-c]quinolin-3-ones represent promising synthetic targets
[3] that have held the attention of researchers in the fields of medicine, pharmacology and
chemistry, for several years. Cook and co-workers, have developed a comprehensive
pharmacophore model for BZDBS at GABAA-R based on structure-activity relationship studies of
136 different ligands from 10 structurally different classes of compounds [20]. The proposed
agonist/antagonist pharmacophore model, based on 2-aryl-pyrazolo[4,3-c]quinolin-3-ones,
3

involves the interaction sites between the receptor and ligand necessary for activity, [21,22]
whereas the structure–activity relationship (SAR and QSAR) findings indicate the importance of
substitutions on the pyrazolo [4,3-c]quinolin-3-one skeleton which determine ligand affinities for
BZDBS at GABAA-R [9, 21,23].
Regarding the tests of anxiety, elevated plus maze test (EPM) is one of the most widely
used animal models for screening anxiolytic and anxiogenic agents. It is an exploratory based
approach–avoidance paradigm measuring non-conditioned responses. This model was validated
by Pellow et al based on the natural aversion of rodents for open spaces and uses an elevated
plus-maze with two open and two closed arms [24,25]. Two indices of anxiety are obtained: the
number of entries into open arms expressed as a percentage of the total number of arm entries,
and the amount of time spent on the open arms expressed as a percentage of the total time on both
open and closed arms. The test is rapid and was found to be sensitive to the effects of both
anxiolytic and anxiogenic agents, anxiolytic agents increasing and anxiogenic agents decreasing
the two measures. Its simplicity and relatively low cost made it essential in the preclinical phase
of industrial research for central nervous system drugs.
The aim of the present study is to report the synthesis of a series of 2-substituted-
pyrazolo[4,3-c]quinolin-3-ones (PQs), the in vitro binding affinities of these compounds to the
BZDBS at GABAA-R through displacement [3H]-Flunitrazepam ([3H]-FNZ) experiments and the
in vivo anxiolytic properties of selected PQs in Wistar adult male rats by Elevated Plus Maze
(EPM).
2. Results and discussion
2.1. Synthesis of pyrazolo[4,3-c]quinolin-3-ones (PQs)
The synthesis of PQs (7-9) were performed through the route outlines in Scheme 1,
according to procedures previously described [26-28] consisting in a Gould-Jacobs modified
4

protocol [5,9,29]. The first step involved the addition of the anilines 1a-d to diethyl 2-
(ethoxymethylene) malonate 2, followed by ethanol elimination, to provide the corresponding
anilinomethylenemalonic esters 3a-d in quantitative yields (> 95%) and in conditions to be used
in the next step without purification (see Scheme 1) [26]. The second step consisted on the
thermal cyclization of 3 in diphenylether (DPE) to give ethyl-4-oxo-1,4-dihydroquinoline-3-
carboxylates 4a-d [5,29,30]. In the next step the chlorination of quinolones 4a-d was carried out
using SOCl2 to furnish the chloro-derivatives 5a-d in a straightforward way. Quinolone
compounds 4a-d and 5a-d were obtained in very good to excellent yields (see Table 1).
Scheme 1. Multistep synthesis of pyrazolo[4,3-c]quinolin-3-ones
5

Table 1. Yields of quinolines 4 and 5
General
Compound
structure
X
R1
R2
R3
Yield (%)a
OH
OH
OH
OH
Cl
Cl
H
H
H
Cl
H
H
H
H
Br
Cl
H
H
H
Cl
H
H
H
78
74
77
58
98
99
98
98
4a
4b
CH3
OCH3
H
4c
4d
5a
Cl
Br
5b
Cl
CH3
OCH3
5c
5d
Cl
aYields of isolated product
In the last step, the reaction of chloroquinolines 5a-d with aryl-hydrazines (6i-iv) and
benzylhydrazine (6v) gave 2-aryl-pyrazoloquinolinones and 2-benzyl-pyrazoloquinolinones,
respectively (see Table 2). In all reactions, anhydrous DMF was used as solvent and cyclization
were completed over 1-4,5 h at 130-140 °C. In the case of the cyclizations with hydrazine
monohydrochloride (6 ii-iv), and dihydrochloride (6v) a previous neutralization with
triethylamine (TEA) was required. The formation of aryl-PQs was good in the most of cases (41-
85%, see Table 2) however, for the synthesis of benzyl-PQs yields were modest taking place the
formation of two isomers (7a,b-v and 8a,b-v in Scheme 1). These findings could be attributed to
the comparable nucleophilicity of both nitrogens in the benzylhydrazine substrate, which allows
the displacement of chlorine by both hydrazinic nitrogens followed by cyclization to give the
6

pyrazolone ring. The ratio of isomers 7a,b-v and 8a,b-v obtained in the reaction mixture depends
on the amount and type of base used in the neutralization step [26].
Table 2. Yields of 2-aryl/benzyl-pyrazolo[4,3-c]quinolin-3-ones 7.
General
Compound
R1
R2
R3
R4
Yield (%)a
structure
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
Ph
p-OMeC6H4
p-FC6H4
Bz
71
41
41
15
61
55
72
10
65
80
60
60
85
73
7a-i
7a-ii
7a-iii
7a-v
7b-i
H
H
Br
Ph
Br
p-OMeC6H4
p-FC6H4
Bz
7b-ii
7b-iii
7b-v
7c-i
Br
Br
CH3
CH3
CH3
CH3
OCH3
OCH3
Ph
p-OMeC6H4
p-FC6H4
o-FC6H4
p-FC6H4
o-FC6H4
7c-ii
7c-iii
7c-iv
7d-iii
7d-iv
aYields of isolated products. BZ refers to benzyl group.
2.2. Specific interaction of PQs with GABAA-R
2.2.1. Determination of [3H]FNZ kinetic binding parameters Bmax and Kd
The kinetics of the [3H]FNZ specific binding to the GABAA-R contained in purified
synaptosomal membranes (SMs) followed a hyperbolic behavior (Fig.1A) characteristic of a
7

single type of binding sites. These values came from the difference calculated between data
obtained in saturation experiments in the presence (nonspecific binding) and absence (total
binding) of a non-labeled displacement agent (Diazepam, DZ) (Fig.1A, inset). The linearity of the
former and its quantitative relation with the latter (less than 25%) reflected the good quality of
the experimental system. The fitness of Eq. 1 (Section 3) to the experimental data in the specific
binding curve, let calculating the kinetic parameters for the [3H]FNZ / GABA-R specific binding,
Kd = 2.3 ± 0.2nM and Bmax = 1702 ± 32 fmol/mg protein (Figure 1A). These values agree with
previous reports [31,32].
2.2.2. Radioligand competition experiment
The potencial GABAergic activity of PQs was tested by investigating their ability to
displace [3H]FNZ from the BDZBS at the GABAA-R through a radioligand competition assay. A
typical experiment performed with compound 7b-ii is depicted in Fig.1B showing a plot of
percent of [3H]FNZ specifically bound in the presence of PQ with respect to the control without
PQ (%B) vs. log PQ concentration (see Supporting Information for a full set of displacement
curves for the other PQs). Fitting Eq. 2 to each displacement plot let determine the relative
binding affinities of non-labelled ligands (IC50) (Eq. 2, section 3). Then, from the IC50 values for
PQs, the corresponding Ki could be calculated according to Eq. 2 [33]. The resulting IC50 and Ki
values (Table 4) showed that, with the exception of dichloro derivatives 7a and the benzyl
derivative 7b-v, PQ compounds were able to displace the [3H]FNZ bound to SM at different
extents.
We found a clear distinction between displacing and not displacing compounds as shown
in the displacement curves depicted in the Supporting Information. Additionally, among the
displacing compounds there was a progression in their potency. The LogIC50 parameter values
were analysed using one-way ANOVA followed by post hoc analysis Tukey to enable specific
group comparisons (P < 0.05 was significant). The ANOVA showed a significant difference
between IC50 values between PQ 7b-ii and 7d-iii, 7d-iv, 7b-iii and 7c-iii; 7c-iv compared with
8

7d-iii, 7d-iv and 7b-iii as well as 7c-ii compared with 7d-iii (see Supporting information for
further details).
1500
1000
6000
4000
2000
500
0
A
B
0
2
4
6
8
10 12
[3H]FNZ (nM)
0
2
4
6
8
10
12
[3H]FNZ [nM]
100
80
60
40
20
0
B
A
-2
-1
0
1
Log [ 7b-ii ] (nM)
Figure 1. Binding of [3H]FNZ at GABAA-R in synaptosomal membranes. A: Typical saturation curve.
The inset shows the TB () and the NB (○). The GABAA-R source was bovine brain cortex SM.
[3H]FNZ and protein concentration were 2 nM and 0.25 mg protein/mL, respectively; B: representative
[3H]FNZ displacement experiment as a function of 7b-ii concentration. Data were fitted to Eq. 2 (inset).
Considering the substituents on the quinoline ring, it was observed that compounds
having two chlorine atoms at positions 7 and 9 (R1 = Cl and R3 = Cl respectively) did not
9

displace the radioactive ligand from the BZDBS, independently of the N-aryl substitution on the
pyrazolone ring. This could be related with a steric and/or electrostatic disturbance area at the
receptor regions described previously as LDi and H2 according to the pharmacophoric model of
Cook and co-workers [20]. Probably the presence of the chlorine atom in position 9 (R1 = Cl)
interferes in the formation of hydrogen bond between the N-1 of the PQ and the H2 receptor site.
In addition, the presence of the two acceptors groups diminish the hydrophobic π-π interaction by
decreasing the electron density on the phenyl ring of the quinolone nucleus, which would reduce
the interaction with the receptor site LDi. In this regard, Savini et al. [6] have tested several
pyrazoloquinolinone compounds as central benzodiazepine receptor ligands and their results of
structure-affinity relationship studies would indicate that the substitution with chlorine at
positions 7 and 9 results unfavourable with respect to the hydrogen substituent.
10

Table 4: Binding affinities of PQs.a
IC50
Ki
PQs
Log IC50 (nM)
(nM)
(nM)
R1
Cl
R2
R3
Cl
R4
Ph
H
-
-
-
7a-i
7a-ii
7a-iii
7a-v
7b-i
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
p-OMeC6H4
p-FC6H4
BZ
-
-
-
-
-
-
H
-
-
-
Br
Ph
-0.084 ± 0.133
0.823
0.326
1.468
-
0.437
0.173
0.780
-
Br
p-OMeC6H4 -0.487 ± 0.137
7b-ii
7b-iii
7b-v
7c-i
Br
p-FC6H4
BZ
0.167 ± 0.065
-
Br
CH3
CH3
CH3
CH3
OCH3
OCH3
Ph
-0.230 ± 0.060
0.589
0.542
1.152
0.370
1.845
1.500
0.313
0.288
0.612
0.196
0.980
0.797
p-OMeC6H4 -0.266 ± 0.040
7c-ii
7c-iii
7c-iv
7d-iii
7d-iv
p-FC6H4
o-FC6H4
p-FC6H4
o-FC6H4
0.062 ± 0.095
-0.433 ± 0.047
0.266 ± 0.057
0.176 ± 0.111
a
The GABAA-R source was bovine synaptosomal membranes, [3H]FNZ and protein
concentration used were 2 nM and 0.25 mg/mL, respectively. The IC50 values were determined
from the % [3H]FNZ bound vs. log PQ concentration (nM) plot, as the PQ concentration that
11

displace the 50% of the [3H]-FNZ bound. Ki was calculated by means of the Cheng and Prusoff
equation (Eq. 2, Ref. 33). BZ refers to benzyl group.
The remaining compounds have displaced 3HFNZ from the BZDBS at the GABAA-R at
different extents according to the substituents at positions 8 and/or the nature of the aryl
substituent at N1, being 7b-ii and 7c-iv two of the most active ligands (Figure 2).
Binding affinities
+
-
Figure 2. Values of IC50 listed in descending order of ligand–receptor binding affinity for PQs 7
The compounds bearing a methyl group at position 8 (R2) have showed very good
affinity, although the compound which has the greatest affinity has a bromine atom in this
position (7b-ii). In this regard, the presence of a methoxy group in C-8 (R2) notably reduces the
affinity of the PQ for this receptor. These results agree with the QSAR relations previously
proposed [6], where the authors have postulated the equation 4, considering the Van der Waals
volume (VW) as an important parameter involved in the biological activity.
pIC50= -0,32 (±0,10 vW) + 9,63 (±0,28)
Eq. 4
n=9, r2=0,883, Q2=0,745, s=0,208, F= 52,77
12

The negative sign of the VW coefficient means that there is a negative steric contribution
for the VW greater substituents. VW values for methyl, bromine and methoxy substituents have
been calculated as 1.01, 1.32 and 1.49 respectively. Considering these values, the methoxy group
promotes the greatest steric effect leading to the increase of the negative character of the equation
term and decreasing the biological activity, as it was observed in the evaluation of PQs 7d.
The examination of the nature of substituents on the phenyl ring attached to N-1 (R4),
indicates that the methoxy group is present in the most active compounds (7b-ii and 7c-ii). The
activity decreases for the 2-phenyl unsubstituted compounds (7b-i and 7c-i) and the behaviour of
fluorine derivatives would depend on the position of fluorine in the phenyl ring. Thus, when the
fluoro substituent is located in the para position of the aryl ring the compounds showed low
activities (7b-iii, 7c-iii and 7d-iii) while the ortho-fluoro phenyl derivatives displayed a
dissimilar behaviour depending on the substitution at C-8. Thus, the 8-methyl-PQ 7c-iv was one
of the most active compounds while the 8-methoxy-PQ 7d-iv showed a reduced activity. In this
regard, Catarzi et al. [34] have proposed that the presence of a halogen atom at C-8 in
combination with a methoxy group at the para-position on phenyl ring produces a steric effect
that improves the interaction of these compounds with the BZDBS at the GABAA-R. Moreover,
Francis et al. [35] have found that the replacement of the H atom at the phenyl ring attached at N-
1 by a methoxy group (ortho and para position), a hydroxy group (para position) or a fluoro
atom increase the compounds affinity. The positive effect of the methoxy group on the para
position of the phenyl ring was also demonstrated in the study of the lead compounds of series
CGS [36]. More specifically, these investigations have indicated that the presence of a fluorine
atom in the ortho position of phenyl substituent improve the affinity of the PQs for the BZDBS
due to the minor size, good electronegativity and the possibility of this halogen to be an acceptor
of H-bond with the H2 region of the receptor [9,34,35]. This behaviour could explain the high
affinity observed for compound 7c-iv.
13

It is noticeable that the derivatives having a benzyl substituent at N-1 (7a-v and 7b-v) did
not show interaction with the BZDBS, probably due to a volume related steric hindrance of this
group limiting the access to the BZDBS cavity in the R-GABAA.
2.3. Elevated Plus Maze experiments
The anxiety-like behaviour of PQs was evaluated in the EPM experiment, which is a
widely used assay for rodents and it has been validated to assess the anti-anxiety effects of
pharmacological agents [24,37]. Rodents are placed in the centre of the four arms of the EPM,
and their behaviour was recorded for 5 min. Anxiogenic drugs reducing time spent on the open
arms and anxiolytic drugs increasing the time spent on the open arms of the EPM.
The brominated PQs 7b-i-iii were tested regarding their anxiolytic properties in Wistar
adult male rats using the EPM experiments. We decided to study these compounds in order to
correlate the results with the structural changes in the PQ nucleus, focussing on the substitution
of phenyl ring attached to N1 (R4). In the Figures 3a-c, the percentage of time in open arms
versus the oral doses provided is shown.
For compound 7b-i, rats treated with doses of 0.25 and 0.5 mg/kg exhibited an increase in
anxiety compared with the rats treated with other doses and vehicle treated control animals
(Figure 3a, ANOVA F(4,28)=3.0521 p=0.0331). These results would indicate an anxiogenic
effect of this PQ at both doses and this behavior could be related to an inverse agonism.
In the experiments performed with 7b-iii, the administration of this compound also
shortened the time spent in the open-sided arms of the plus-maze (Figure 3b). The effect
gradually increased with the increment on doses with the peak of the effect observed at 3 mg/kg
(ANOVA F(4,28)=4.6784 p=0.0062). It is worthy to note that a great reduction of entries of
animals to the closed arms was evidenced, which could reflect a reduced capacity of locomotion
14

(ANOVA F(4,24)=4.7678 p=0.0056). In this case, it is difficult to attribute the reduced
permanence in open arms to a purely anxiogenic effect because significant changes in motor
activity were observed. Further studies are needed to determine the exact mechanism for this
effect which might be due to a sedative or toxic action of this compound.
The administration of 7b-ii resulted in a significant increase in the time spent in the open-
sided arms, with the peak anxiolytic-like effect observed at 0.50 mg/kg (Figure 3c ANOVA
F(4,26)=7.5553 p=0.0004). In this case, the motor activity of animals was not suppressed at any
evaluated doses (ANOVA F(4,25)=1.2986 p=02975).
Considering that the methoxy derivative 7b-ii presented a remarkable anxiolytic effect, the
EPM experiments with the PQ 7c-ii (Figure 3d) were carried out to compare the effect of
substituent in C-8 keeping the p-methoxy group in the N-phenyl ring. In this case, 7c-ii caused an
increment in the time spent in the open-sided arms (50%) at 1mg/kg compared with a 20%
response rate in vehicle treated control animals (ANOVA F(4,24)= 5.0932 p=0.0040) without
any significant effect on the locomotor activity (ANOVA F(4,24)=1,1750 p=0.3467). Although
the administration of 7c-ii was effective in producing an anxiolytic-like effect, the doses were
two times the required for the same effect with the compound 7b-ii. Preliminary, these PQs could
be an agonist in the BZDBS at GABAA-R.
15

(a)
(b)
(c)
(d)
Figure 3. Elevated plus maze tests for PQs 7b-i (a), 7b-ii (b), 7b-iii (c) and 7c-ii (d). Data
represent the mean ± SEM of the percentage of time spent on open arms relative to total time
spent on all four arms. ⁎p≤ 0.05 vs. other groups (n=6-8).
2.4. Principal component Analysis.
Principal component analysis (PCA) is a multivariate type analysis here applied to
ascertain which molecular property or properties combination of PQs were associated with the
differential behavior of these compounds on displacement and EPM experiments (see Table S33,
in Supporting Information). The contribution of a molecular parameter or effect to each Principal
Component (PC) is reflected by the loading value (eigenvalue) derived from PCA analysis (see
Table S34, in Supporting Information).
The PCA graphical representation is shown in Figure 4 (data taken from Table S35,
Supporting Information). The first two PCs explained 56% of total variance. The first principal
component (PC1) explained 42% of total variance and was mainly influenced (loading values
16

|0.39|) by the variables 1 (R1-Cl), 3 (R2-H), 7 (R3-Cl), 11 (R4-BZ), 14 (IC50). PC1 separated the
PQs unable to displace FNZ from GABAA-RBS (bearing a chloride atom at positions 7 (R1) and 9
(R3) from the rest of PQs studied. The second principal component (PC2, which explained 14%
of total variance), was mainly influenced by the variables 5 (R2-CH3), 10 (R4-p-OMeC6H4), 15
(EPM-tOA) and separated the PQs that induced an increase or decrease in the time spent on the
open arms.
Figure 4. Graphical representation of the Principal Component Analysis. The first two principal
components were plotted in a phase space where the grouping of related variables become clear. Data
points are represented by the same identification numbers given to variables in Table S1 (Supporting
Information). Red circles identify PQs with variables exhibiting negative and positive loadings for PC1
and associated with high and low IC50 values, respectively. Blue circles identify PQs with variables
exhibiting positive and negative loadings for PC2 and associated with high and low EPM-tOA values,
respectively.
17

3. Materials and methods
3.1. Chemistry
Reagents and solvents were used from commercial sources without purification. Melting
points were determined by using a Electrothermal 9100 capillary melting point apparatus and are
uncorrected. 1D- and 2D NMR spectra (1H, 13C, ROESY, HSQC, HMBC) were recorded at room
1
temperature on a BRUKER FT-400 MHz spectrometer at 400 MHz for H and 100 MHz for 13C,
using the indicated solvents. Chemical shifts are given in parts per million (ppm) relative to
central peak of the solvents. Coupling constants values are given in Hertz. Mass spectra were
recorded on a SHIMATZU CGMS-QP 5050A system operating at 70 e.v. High-resolution mass
spectra were recorded with an Agilent LCTOF instrument.
3.1.1. General procedure for the preparation of pyrazoloquinolinones 7a-d
The pyrazoloquinolinone compounds were prepared following a protocol previously
described [26]. Equimolar amounts (3 mmol) of aniline and diethyl(ethoxymethylene)malonate
were mixed and heated at 80°C, during 1h to give malonates 3a-d, which were cyclized in
diphenyl ether at 220-230°C during 1-5 h to afford quinoline derivatives 4a-d. These quinolones
(0.8 mmol) were mixed with excess of thionyl chloride (0.5 mL) at reflux temperature for 1-3 h
to give chloroquinolines 5a-d. Pyrazoloquinolinones 7a-d were prepared by reaction between 5a-
d (0.8 mmol) and the hydrochloride salt of hydrazines 6i-v (1 mmol), previously neutralized with
triethylamine, using dimethyl formamide (DMF) as solvent and temperatures between 130-
140°C. Compounds 7b-i, 7c-iv and 7d-iii were recrystallized from DMF, ethanol/water and
DMF/water respectively; 7b-ii, 7b-iii, 7c-ii, 7c-iii and 7d-iv were purified by chromatography
column and 7c-ii was obtained as pure compound. All PQ compounds were characterized by
18

standard spectroscopic techniques (1H NMR, 13C NMR, HMBC, HSQC) and high-resolution
mass spectrometry, and all data agree with the proposed structures, which have been previously
reported [5,6,26-29,38].
3.2. Biochemical Experimental Section
3.2.1. In vitro binding experiments
3.2.1.1. Materials
The BZD diazepam (DZ) was kindly supplied by Products La Roche (Córdoba,
Argentina). [3H]-FNZ was purchased from New England Nuclear Chemistry (E.I. DuPont de
Nemours & Co. Inc., Boston, MA, USA). Other drugs and solvents were of analytical grade.
3.2.1.2. Natural membrane preparation
Synaptosomal membranes (SM) were obtained from bovine brain cortex. Meninges were
eliminated, the cortex dissected, and the SM were purified essentially according to the method of
Enna and Snyder, modified by Perillo and Arce [39] immediately lyophilized and stored at -20ºC.
Immediately before use, membranes were resuspended in 50 mM pH 7.4 Hepes buffer containing
100 mM NaCl at a final protein concentration of 0.25 mg/mL. This SM suspension was used as
membrane receptor preparation and GABAA-R source in the experiments that followed.
3.2.1.3 Radioligand binding assays.
3.2.1.3.1 Saturation Curve
All the procedures were carried out at 4ºC. The membrane receptor preparation was
obtained as described above. The BZDs used as labelled and non-labelled ligands were [3H]FNZ
and DZ, respectively. Both BZDs compete for their reversible interaction with the same binding
site at GABAA-R (specific interaction). On the other hand, due to their lipophilic characteristics,
BZDs interact, through hydrophobic interactions, with the GABAA-R containing membrane by an
19

irreversible partitioning process (nonspecific binding, NB). So, only the specific binding of
[3H]FNZ can be displaced from the receptor with high concentrations of the (DZ) and the
remaining non-specifically bound [3H]FNZ represents NS. TB (total binding) is measured in the
presence of the same concentration of [3H]FNZ but in the absence of non-labeled ligand.
The incubation system (230 mL total volume) contained: the SM suspension at a final
0.25 mg/mL total protein concentration of and 0.5–12 nM [3H]FNZ (minimum specific activity
81.4 Ci/mmol). Then, 100 mM NaCl–50 mM Tris–HCl pH 7.4 buffer, containing (NB samples
set) or not (TB samples set) 9.4 mM DZ. Samples were incubated at 4 °C in the dark for 1 h and
then filtered through SS filters Whatman GF/B type with a Brandel automatic filtration apparatus
(Brandel, Gaithersburg, MD, USA). Then, filters were rinsed, dried in the air stream and placed
in vials containing 2.5 mL of scintillation liquid (25% v/v Triton X-100, 0.3% w/v
diphenyloxazole in toluene). The retained radioactivity was measured with a scintillation
spectrometer Rackbeta 1214 (Pharmacia-LKB, Finland) at a 60% efficiency for tritium. Each
sample was run at least in duplicate determinations.
Specific binding (B) was calculated as the difference between TB and NB determined in
the absence and in the presence of DZ, respectively. Eq. 1 was fitted to the saturation curves
(specific binding (B) versus free [3H]FNZ concentration), by a non-linear regression analysis
performed by a computer-aided least squares method [39].
Eq. 1
Bmax .F
B 
Kd  F
where F is the free [3H]FNZ concentrations, B and Bmax are the ligand concentration-dependent
and the maximal specific binding activities, respectively, and Kd is the equilibrium dissociation
constant. Protein concentration was determined by the method of Lowry [40].
20

3.2.1.3.2 [3H]FNZ displacement curves
The experiments were carried out using a 2nM constant radioligand ([3H]-FNZ)
concentration corresponding to the mean dissociation binding constant (Kd) determined in a
previous experiment. The SM (0.25 mg prot/mL) was incubated with the radioligand and with
increasing concentrations of unlabelled PQ at 4°C, in the dark for 1 h. After incubation, samples
were filtered through SS filters Whatman GF/B type with an automatic harvester apparatus. The
filters rinsed and dried in the air, were placed in vials containing 2.5 ml of scintillation liquid
25%V/V Triton X-100, 0.3% P/V diphenyloxazole in toluene. The retained radioactivity was
determined with a scintillation counter Rackbeta 1214 (Pharmacia-LKB, Finland) with an
efficiency of 60% for Tritium. Protein concentration was determined by the method of Lowry
[40]. Each sample was run at least in duplicate determinations.
The IC50 values were determined from the % [3H]-FNZ bound (%B) vs. log PQ
concentration (nM) plot. If a compound competes for a homogeneous class of binding sites, the
competition curve follow a sigmoidal function with a Hill-slope (H) equals unity and the data
points fit to a 4-parameter logistic equation (Eq. 2), were Bbottom is the %B in absence of PQ and
Bbottom is the %B at the highest PQ concentration assayed. The IC50 value is obtained from the
curve inflection point the and represent the PQ concentration that displace the 50% of the [3H]-
FNZ bound.
Eq. 2
Then, if the Kd of the labeled ligand is known, Ki value can be calculated by means of the Cheng
and Prusoff equation (Eq.3) [39].
Ki=IC50/{1+[radioligand]/Kd}
Eq. 3
21

Although IC50 is a very informative parameter, Ki values have the advantage over IC50 of being
independent on the radioligand concentration used.
3.2.1.3.3 Protein quantitation
Protein concentration was quantified according to the Lowry method [40]. Briefly, the
system consisted of 0.05ml of SM suspension and 0.5ml of EDTA-Cu2+. The samples were
incubated at room temperature by 45 min and 0.05 mL of 1:2 bidistilled water diluted Folin
Ciocalteu reagent was added. The samples were incubated more 1 h at room temperature. Finally,
the absorbance at 750 nm was registered using a Beckman DU 7500 spectrophotometers 0.0001
AU sensibility.
3.3. In vivo behavioural test
3.3.1 Animals
Adult male Wistar rats (65–75 days), bred in our colony and weighing 280–320 g, were
housed in groups of 4-5 per cage with food and water ad libitum. They were maintained in a 12 h
light–dark cycle (lights on at 07:00 a.m.) at a constant room temperature of 21-22 °C. Rats were
handled during the week before the experimental procedure, in order to habituate them to
manipulation. This habituation consisted in the transportation of the animals to an experimental
room, removing them from their cages, the handling of each animal during 1min and returning
them to their home cages. This procedure was repeated during four consecutive days before the
experiments. All the experiments were performed during the light cycle between 10:00 a.m. and
03:00 p.m.). An average of 7 rats was used for each dose for each compound. Procedures were
conducted in accordance with the National Institutes Health Guide for the Care and Use of
Laboratory Animals, as approved by the Animal Care and Use Committee of the Faculty of
Chemical Sciences, National University of Córdoba, and by the National Department of Animal
22

Care and Health (SENASA-ARGENTINA). Efforts were made to minimize animal suffering and
to reduce the number of animals used.
3.3.2 Elevated plus-maze (EPM)
The apparatus consisted of a black acrylic maze with two opposite open arms (50×10 cm)
and two opposite closed arms (50×10×40 cm) disposed like a plus sign with a central square of
10 cm2. Entire maze was elevated 50 cm above the floor. The test consisted in placing the rat in
the central square of the maze facing one closed arm and allowing it to explore during 5 min.
This procedure was performed in a quiet and dimly illuminated room (4 lx). The scores analysed
were the time spent in open and closed arms, and the number of entries to both arms. With these
data were calculated the percentage of time spent in open arms which is widely used as an
anxiety index (time spent in open arms relative to the time spent in open and closed arms). The
number of entries in the closed arms was also assessed, as indicative of exploratory activity [41-
44].
3.3.3 Formulation and administration of compounds
Each drug was dissolved in propylenglycol by sonication and then diluted with an
aqueous solution of 0.6 % v/v Tween 80. The dose tested were 0.25, 0.5, 1 y 3 mg/kg. These
solutions were prepared the same day of the experiment. Each rat was injected intraperitoneally
30 min before the exposition to the behavioral test (EPM). The control rats were injected with a
solution of propylenglycol/ Tween 80/ water in the same proportion that used to dissolve the
drugs.
3.3.4 Statistical analysis
23

The data were analysed statistically using the software Statistic 6.0. The values graphed
represent the means ± S.E.M (standard error of the mean). The EPM data and LogIC50 parameter
values were analysed using one-way ANOVA followed by post hoc analysis (Fischer LSD to
EPM) to enable specific group comparisons or by post hoc analysis Tukey (to LogIC50 data) [45],
P < 0.05 was significant. Principal component analysis (PCA) was performed on the molecular
properties, binding data and EPM data, previously centered and standardized since they were
expressed in non-comparable units [46] using statistical InfoStat software [47].
4. Conclusions
Pyrazoloquinolinones 7a-d and were prepared and tested as central benzodiazepine receptor
ligands. The substitution on quinoline ring as well as the nature of substituent on N-2 was
decisive for the affinity for GABAA-R in the in vitro displacement experiments. The presence of
chlorine in positions 7 and 9 of the quinoline moiety or the presence of a benzyl group attached at
the N-2 of the pyrazolone ring led to a lack of ability of PQs to displace [3H]FNZ binding from
bovine brain membranes. However, PQs bearing a methyl group at C-8 of quinoline ring and p-
methoxy phenyl group (7c-ii) or o-fluoro phenyl group (7c-iv) at N-2 showed a remarkable
activity with IC50 (nM) values of 0.542 and 0.370 respectively. Also, the PQ having a bromine
substituent at C-8 and a p-methoxy phenyl group at N-2 (7b-ii) showed high selective affinity for
the BZD binding site at GABAA-R, with IC50 (nM) value of 0,326.
In the EPM test PQs 7b-ii and 7c-ii, both compounds having a p-methoxy phenyl group at
N-2 and high affinity for the GABAA-R, presented a significant anxiolytic effect with the dose of
0.5 and 1.0 mg/kg. These results suggest a possible role of these compounds as agonists of the
benzodiazepine binding site. Also, this behaviour would indicate that PQs were well absorbed
and crossed the blood-brain barrier.
24

Acknowledgements
This study was financially supported by grants from CONICET and SECyT-Universidad
Nacional de Córdoba.
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28

GRAPHICAL ABSTRACT
Relative binding affinityto BZD binding site at GABA -R
A
[3H]FNZ binding site
GABAA-R
Anxiogenics
100
80
60
40
20
0
Anxiolytics
IC50 0,33 nM
Ki 0,17 nM
Plus Maze Test
3
[
H]FNZ displacement
-2
-1
- Log 17f (nM)
0
1
Log PQ 7b-ii [nM]
29