Anxiolytic-like effect of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol is mediated through the benzodiazepine and nicotinic pathways

Article abstract of DOI:10.1111/cbdd.12961

In this study, we proposed the design, synthesis of a new compound 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032), and pharmacological evaluation of its anxiolytic-like effect. This new compound was subjected to pharmacological screening referred to as Irwin test, prior to sodium pentobarbital-induced sleep, open-field and wire tests. The anxiolytic-like effect of this compound was evaluated using elevated plus maze and light–dark box tests. In addition, the mnemonic activity was evaluated through step-down test. In sodium pentobarbital-induced sleep test, LQFM032 decreased latency and increased duration of sleep. In the open-field test, LQFM032 altered behavioral parameter, that suggested anxiolytic-like activity, as increased in crossings and time spent at the center of open field. In the plus maze test and light–dark box test, the LQFM032 showed anxiolytic-like activity, increased entries and time spent on open arms, and increased in number of transitions and time spent on light area, respectively. Those effects was antagonized by flumazenil but not with 1-(2-Methoxyphenyl)-4-(4-phthalimidobutyl)piperazine (NAN-190). The LQFM032 did not alter mnemonic activity. Moreover, the anxiolytic-like activity of LQFM032 was antagonized by mecamylamine. In summary, LQFM032 showed benzodiazepine and nicotinic pathways mediated anxiolytic-like activity without altering the mnemonic activity.

Full text of DOI:10.1111/cbdd.12961

PROF. ELSON ALVES COSTA (Orcid ID : 0000-0003-1996-0901)
Received Date : 30-Sep-2016
Revised Date : 17-Jan-2017
Accepted Date : 21-Jan-2017
Article type : Research Article
ANXIOLYTIC-LIKE EFFECT OF 2-(4-((1-PHENYL-1H-PYRAZOL-4-
YL)METHYL)PIPERAZIN-1-YL)ETHAN-1-OL IS MEDIATED THROUGH THE
BENZODIAZEPINE AND NICOTINIC PATHWAYS
1Adriane F. Brito, 1James O. Fajemiroye, 1Hiasmin F.S. Neri,1Dayane M. Silva, 1Daiany P.B
Silva; 3Germán Sanz, 3Boniek G. Vaz, 4Flávio S. de Carvalho, 1Paulo C Ghedini, 4Luciano M.
Lião, 2Ricardo Menegatti, 1Elson A. Costa
1Department of Pharmacology, ICB, Federal University of Goiás, Campus Samambaia, 74001-970,
314, Goiânia, GO, Brazil
2Faculty of Pharmacy, Laboratory of Medicinal Pharmaceutical Chemistry, Federal University of
Goiás, Goiânia, GO, Brazil
3Chemistry Institute, Laboratory of Chromatography and Mass Spectrometry, Federal University of
Goiás, Goiânia, GO, Brazil
4Chemistry Institute, Federal University of Goias, Campus Samambaia, Goiânia, GO, Brazil
Corresponding author
xico@ufg.br
ABSTRACT
In this study we proposed the design, synthesis of a new compound 2-(4-((1-phenyl-1H-
pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032), and pharmacological evaluation
of its anxiolytic-like effect. This new compound was subjected to pharmacological screening
refered to as Irwin test, prior to sodium pentobarbital-induced sleep, open field and wire tests.
The anxiolytic-like effect of this compound was evaluated using elevated plus maze and light-
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dark box tests. In addition, the mnemonic activity was evaluated through step-down test. In
sodium pentobarbital-induced sleep test, LQFM032 decreased latency and increased duration
of sleep. In the open field test, LQFM032 altered behavioral parameter, that sugested
anxiolytic-like activity, as increased in crossings and time spente at the center of open field. In
the plus maze-test and light-dark box test, the LQFM032 showed anxiolytic-like activity,
increased entries and time spent on open arms, and increased in number of transitions and
time spent on light area, respectively. Those effects was antagonized by flumazenil but not
with 1-(2-Methoxyphenyl)-4-(4-phthalimidobutyl)piperazine (NAN-190). The LQFM032 did
not alter mnemonic activity. Moreover, the anxiolytic-like activity of LQFM032 was
antagonized by mecamilamine. In summary, LQFM032 showed benzodiazepine and nicotinic
pathways mediated anxiolytic-like activity without altering the mnemonic activity.
Keywords: benzodiazepine site, nicotinic activity, piperazine derivative, LQFM032.
1.0. INTRODUCTION
Diseases that affect mental health are common, for example, in the US in 2014 it was
estimated that 43.6 million adults in US (18.1% of Americans) had mental disorder [1, 2, 3, 4, 5]
Mental disorders are associated with social-economical problems and high annual medical
cost that exceeds US $ 317 billion [3, 4]
.
.
Mental disorders are generally characterized by changes in mood, thinking and/or
behavior. Nowadays, anxiety is among the most leading mental disorders [5]. Although
anxiety could be beneficial (in a physiological situation), excessive anxiety could lead to
suffering and affect daily activities. Symptoms such as difficulty concentrating, irritability,
muscle tension and trouble sleeping are commonly found among anxiety patients [1, 6]
.
Currently, anxiety is being treated with different classes of anxiolytics drugs.
Benzodiazepine such as diazepam and clonazepam are effective agents but their clinical
[7, 8]
application is associated with tolerance, the risk of abuse, anterograde amnesia
among
other undesirable effects. For this reasons, the benzodiazepines are not considered as the first
choice of treatment[1, 9]. The selective serotonin reuptake inhibitors, and dual selective
inhibitors of the reuptake of serotonin and norepinephrine, seems to be prefered as the first
line of therapy[9]. However, it takes weeks of consistent administration of these drugs to
achieve anxiolytic effects. Hence, further research with the aime of developing effective
treatments with fewer side effects becomes a necessity[10].
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In the current study, we describe the biological evaluation of a new heterocyclic
piperazine derivative, LQFM 032 (4), which was designed from hybridization molecular
strategy from LQFM008 (2) and JWB-1-84-1 (3) - lead compounds. LQFM 008 (2) is an
anxiolytic and antidepressive-like compound[11, 12] that was synthesized through molecular
simplification of clozapine (1), while JWB-1-84-1 (3) is an analogue of acetylcholine with
neuroprotective properties [13]. As illustrated in Figure 1A, we changed ethyl 1-
peperazinacarboxylate C by 1-(2-hydroxyethyl)piperazine C.
2. Material and Methods
2.1. Chemical synthesis
2.1.1. General methods
Reactions were monitored by TLC using commercially available precoated plates
(Whatman 60 F254 silica) and developed plates were examined under UV light (254 and 365
1
13
nm). H and C NMR spectra were recorded in the indicated solvent on Bruker Avance III
500 MHz spectrometer. Chemical shifts were quoted in parts per million downfield from
TMS and the coupling constants in Hertz. Infrared spectra were recorded on a Perkin- Elmer
Spectrum Bx-II FT-IR System spectrophotometer instrument as films on KBr discs. Melting
points were performed using a Marte melting point apparatus, and the results were
uncorrected. All assignments of the signals of 1H and 13C NMR spectra are consistent with the
chemical structures of the products described. The organic solutions were dried over
anhydrous sodium sulfate and organic solvents were removed under reduced pressure in a
rotary evaporator. Mass spectra (MS) were obtained with a QQ-TOF (Brucker Daltonics
Bremen, Germany). The sample preparation for MS analysis consisted of diluting 1 mg of
each sample in 1 mL of methanol. In order to perform the analysis in positive and negative
mode, 1 μL of formic acid and 1μL of ammonium hydroxide, respectively, were added to the
samples. The solution obtained was directly infused at a flow rate of 2μL/min into the ESI
source. The ESI ( ) source conditions were as follows: a nebulizer gas pressure of 0.5 - 1.0
bar, a capillary voltage of 3.0 kV and a transfer capillary temperature of 250 °C.
2.1.2. Synthesis of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanol (4)
5 mL of MeOH was added in ZnCl2 (0.5 mmol). After was added this compounds in
aldehyde (6) (1.0 mmol), that contained 1-(2-hydroxyethyl)piperazine (7) (1.0 mmol) and
NaBH3CN (0.5 mmol) (Figure 1B). The mixture was stirred at reflux temperature for 2
hours[14]. In tu rn, MeOH was then evaporated and the residue was partitioned between water
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and CH2Cl2. The combined organic layers were dried (Na2SO4), concentrated in vacuum, and
the crude product was purified through chromatography method with hexane:ethyl acetate as
mobile phase to provide 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanol (4)
as a beige oil in 73.0% of yield, Rf=0.38 (dichloromethane/methanol, 90:10): IRmax (KBr)
cm−1: 3392 (v O-H), 1559 (v C=C aromatic) and 1402 (v CH2) (Figure 2A); 1H-NMR (500.13
MHz Figure 2B) CDCl3 δ: 7.90 (1H, s, H-5), 7.67 (2H, m, H-2’ and H-6’), 7.64 (1H, s, H-3),
7.44 (2H, m, H-3’ and H-5’), 7.27 (1H, m, H-4’), 3.66 (2H, t J=5.28, H-8’’), 3.54 (2H, s, H-
6), 2.98 (1H, s, H-9’’), 2.67 (8H, m, H-2’’, H-3’’, H-5’’ and H-6’’), 2.62 (2H, t, J=5.28, H-
7’’); 13C-NMR (125.76 MHz – Figure 2C) CDCl3 δ: 141.8 (C-3), 140.0 (C-1’), 129.4 (C-3’
and C-5’), 126.7 (C-4’), 126.4 (C-5), 118.9 (C-2’ and C-6’), 118.8 (C-4), 59.4 (C-7’’), 57.6
(C-8’’), 52.8 (C-2’’, C-3’’, C-5’’ and C-6’’), 52.3 (C-6). MS: [M+ H]+m/z of 287.189 (Figure
2D).
2.2. Pharmacological evaluation
2.2.1. Drugs and treatments
The control group received polisobarte 80 2% (vehicle – Tween 80 – Synth, USA) at
the dose of 10 mL/Kg. LQFM032 (2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-
yl)ethan-1-ol – Molecular Weight - MW: 286.18 mg/mmol) was administered by routes p.o.,
s.c. and i.p., at the doses between 7.0 - 875 µmol/Kg for Irwin test. In specific tests, like open
field and plus maze, LQFM032 was administered orally at doses of 18, 54 and 162 µmol/Kg.
LQFM032 was dissolved in Tween 80 2%. Sodium pentobarbital (MW: 226.26 mg/mmol,
Sigma Chemical Co., USA) was administred at the dose of 220 µmol/Kg. Flumazenil (MW:
303.29 mmol/mg, União Química, Brazil), benzodiazepine site competitive antagonist, was
administred at the dose of 6.6 µmol/Kg i.p., 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)
buthyl] piperazine hydromide (NAN-190 – MW: 393.48 mg/mmol, Chemical Co., USA), 5-
HT1A competitive antagonist, was administred at the dose of 1.3 µmol/Kg i.p. To verify the
pharmacological effects of benzodiazepine site and 5-HT1A receptor agonists, diazepam
(MW: 284.73 mg/mmol, Cristália, Brazil) at a dose of 3.51 µmol/Kg p.o. and buspirone (MW:
385.50 mg/mmol, Sigma, USA) at a dose of 26 µmol/Kg p.o. Scopolamine (MW: 303.34
mmg/mmol, Sigma, USA), muscarinic receptor antagonist was administered at dose of 1.0
µmol/Kg i.p. to induce memory.
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2.2.2. Animals
Adult male Swiss mice weighing approximately 30 g, six weeks old, were used in all
experiments. These animals were provided by the Central Animal House of Federal
University of Goiás (UFG). All experimental groups, except for Irwin test, had eight mice per
group. The animals were housed in groups of 20 mice/cage (50 x 23 x 16 cm) and were
maintained under controlled conditions of temperature (25 2ºC) with a 12 h dark/light cycle,
with food and water ad libitium. The tests were conducted between 8:00 a.m. and 4:00 p.m.
All experiments were carried out according to the Ethical Principles in Animal Research as
adopted by the Brazilian Society of Laboratory Animal Science and approved by the Ethics
Committee on Animal Use of University Federal of Goiás-Brasil (nº 023/13).
2.2.3. Effects on gross behavior or Irwin test
Experimental groups of mice (n=3 per group) were treated orally (p.o.),
intraperitoneally (i.p.) or subcutaneously (s.c.) with LQFM032 at doses of 7, 35, 175 and 875
µmol/Kg whereas control group received vehicle 10 mL/Kg by the same routes. The animals
were observed in free ambulation on a flat surface for 3 min, at 0, 5, 10, 20, 30 and 60 min, 4,
8, 24 and 48 h, 4 and 7 days after the treatment. The observed effects were noted using a
standard pharmacological screening approach [15]
.
2.2.4. Sodium pentobarbital-induced sleep test
Experimental groups of 8 mice each were treated orally with vehicle 10 mL/Kg or
LQFM032 at doses of 18, 54 and 162 µmol/Kg. Sixty minutes after the treatment, the animals
received sodium pentobarbital (220 µmol/Kg, i.p.). Latency to induce sleep (loss of the
rigthing reflex) and the duration of the sleep (the time required to recover the righting reflex)
were recorded for each animal [16]
.
2.2.5. Open field test
The apparatus consisted of a circular arena measuring 36 cm (diameter) x 20 cm
(height), with the floor divided into eight squares of equal area. Mice (n=8) were individually
placed at the center of the open field apparatus to measure during 5 min locomotor activity
after 60 min of oral treatment with vehicle or LQFM032 (18, 54 and 162 µmol/Kg). The
number of squares crossed, rearing behaviors, immobility time (s), the number of crossings
and time (s) spent at the center (%CrCe and TCE) of the arena were recorded [17, 18]
.
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2.2.5. Wire hanging test
Groups of mice (n=8) were treated orally with vehicle or LQFM032 (18, 54 and 162
µmol/Kg). One hour after treatment, animals were suspended from an elevated wire by their
forepaws at a height of ~20 cm above the floor to prevent the animal from climbing down.
The animal was placed at the center of the wire. The time that elapsed until the animal fell
was recorded three times and the cutoff time was set at 60 s. The latency to the falls was
recorded and analyzed [16]
.
2.2.6. Elevated plus maze test (EPM)
The EPM apparatus consisted of two open arms (30 cm x 5 cm x 0.5 cm) and two
closed arms (30 cm x 5 cm x 15 cm) connected by a commom central platform (5 cm x 5 cm).
The experimental groups of 8 mice each that were treated p.o. with vehicle or LQFM032 (18,
54 and 162 µmol/Kg) were exposed to EPM apparatus. Sixty minutes after the treatment,
mice were placed individually at the center of the EPM facing one of the enclosed arms and
observed for 5 min [19, 20]. The test was carried out under a red light (15 W) and was fully
recorded for later analysis. Parameters such as number of entries (EOA) and the time spent in
the open arms (TOA) of EPM were used as a measure of anxiety. Anxiolytic compounds
reduce the animal’s aversion to the open arms and promote its exploration.
2.2.7. Light-dark box test (LDB)
Experimental groups (n=8) mice were treated p.o. with vehicle or LQFM032 (18, 54
and 162 µmol/Kg). Sixty minutes after the treatment animals were placed at the center of the
light area (20 x 26 x 26 cm) facing the opening (7 cm x 7 cm) of the dark area (20 cm x 26 cm
x 17 cm). The number of transitions between the compartiments and the time spent in the
light area (TLA) were recorded over a 5 min [21]
.
2.2.8. Mechanism of anxiolytic like property
In order to investigate the possible mechanisms underlying the anxiolytic-like
activities of LQFM032, the animals were pre-treated i.p., with saline 0.9% (10 mL/Kg),
flumazenil (6.6 µmol/Kg) or NAN-190 (1.3 µmol/Kg). After 30 min, the animals were treated
p.o. with vehicle, LQFM032 (54 µmol/Kg), diazepam (3.51 µmol/Kg) or buspirone (26
µmol/Kg). Sixty minutes after the last treatment, mice were submited to the light-dark box
test.
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2.2.9. Effects of LQFM032 on avoidance memory in stepdown test
The stepdown avoidance test was used to evaluate the possible effect of LQFM032 on
cognition. The animals were submitted to one training session, after 60 min of treatment p.o.
with vehicle or LQFM032 (18, 54 or 158 µmol/Kg, p.o.). The mice were placed on the
platform and the stepdown from the platform with all four paws was immediately followed by
a foot-shock (0.5 mA) for 1 s. The latency to stepdown was recorded. Afteward, the animals
were exposed to the apparatus again (test session) after an interval of 90 min and 24 h from
the first exposure, training session (TS). The test session was conducted in the absence of
shock and the stepdown latency from the platform was recorded (up to 180 s) and evaluated
as indicative of memory retention [22]
.
In order to evaluate the capacity of LQFM032 to prevent scopolamine-induced
memory deficits, the animals were pre-treated p.o., 30 min before, with LQFM032 (18
µmol/Kg) or vehicle. Then, animals were treated i.p. with saline or scopolamine 1.0 µmol/Kg.
Thirty minutes after the last treatment, the animals were exposed to TS, and the the latency to
step-down (s) was recorded.
2.2.10 Evaluation of nicotinic receptor participation in anxiolytic-like activity of LQFM032
In order to verify the involvement of nicotinic receptor in the anxiolytic-like activity
of LQFM032, the animals were pre-treated i.p. with saline or mecamylamine 30 µmol/Kg.
Then, 30 min after, were administred p.o. vehicle or LQFM032 54 µmol/Kg. One hour after
the last treatment, LDB test was realized and the time spent in the light area and number of
transitions between compartments were recorded.
2.2.11 Statistical analysis
Results were expressed as means standard error of mean (SEM). Data was analyzed by
one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls as the post-hoc
test. In the wire hanging test, the data were analyzed by the Kruskal-Wallis test followed by
Dunns as post hoc test and expressed as median (25th percentile to 75th percentile). Effects
were considered significant at p≤0.05. All graph was drawn using GraphPad Prism version
5.0® software.
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3.0. Results
3.1. Synthesis of LQFM032
As illustrated in Figure 1B, the synthetic route began with 1-(phenyl)-1H-pyrazole (5)
and proceeded through the classical method described by Finar and Godfrey, in 88% of yield
(Finar, 1957). Chemoselective and regiospecific formylation of 1-(phenyl)-1H-pyrazole (5) to
1-(phenyl)-1Hpyrazole-4-carbaldehyde (6) was performed under Duff’s conditions in 83% of
yield (de Oliveira, 2013). The synthesis of 2-(4-((1-phenyl-1H-pyrazol-4-yl) methyl)
piperazin-1-yl) ethanol (4) was carried out under conditions of reductive amination in 73 % of
yield (Lou, 2004). LQFM032 (4) was obtained in three synthetic steps with 53 % overall
yield.
3.2. Effects on gross behavior or Irwin test
Animals treated with LQFM032 at the dose of 7 µmol/Kg (p.o., i.p. or s.c.) did not
altered behavioral activity. However, LQFM032 at dose of 35 µmol/Kg (s.c.) decreased
exploratory activity after 5 minutes of administration. Treatment at dose of 175 µmol/Kg
(s.c.) induced ataxia and lethargy. The same dose through i.p. or p.o. increased exploratory
activity. Finally, at dose of 875 µmol/Kg (p.o., i.p. or s.c.) behavioral parameters such as
sedation, diarrhea, paralysis of the hindlimbs were observed. All behavioral alterations
(induced by LQFM032 treatment at diferents doses and routes) ceased after 4 days without
any death record at the end of the 7 days of observation.
3.3. Sodium pentobarbital-induced sleep test
Treatment with LQFM032 54 and 162 µmol/Kg decreased sleep latency and increased
sleep time (Figure 3).
3.4. Open field test
The treatment with LQFM032 at dose of 162 µmol/Kg decreased number of crossings
and rearings, while the immobility time was not changed significantly. At the dose of 54
µmol/Kg, LQFM032 did not alter number of crossings and immobility time, but decreased
number of rearings. The treatment of different doses of LQFM032 increased the time spent
(TCe) and crossings at the central (%CrCe) area (Table 1).
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3.5. Wire Hanging Test
LQFM032 at differents doses did not alter the values of latency off fall as represented
by the group median (Table 1).
3.6. Elevated plus maze test (EPM)
The treatments with different doses of LQFM032, increased the percentage of entries
and the time spent in open arms (Figure 4).
3.7. Light-Dark box test (LDB)
Treatment with LQFM032 at doses of 54 and 162 µmol/Kg increased the number of
transitions between the two compartments (light/dark). Moreover, all the doses of LQFM032
increased time spent in light area (Figure 5).
3.8. Mechanism of anxiolytic like property
The anxiolytic-like activity of LQFM032 was antagonized by flumazenil. However,
pretreatment with NAN-190 did not reversed this activity, demonstrated by number of
transitions (Figure 6A) and time spent in light area (Figure 7A) of LDB.
3.9. Effects of LQFM032 on avoidance memory in stepdown test
LQFM032 (18, 54 and 162 µmol/Kg) did not cause memory impairment on short- and
long-term memory (after 90 min and 24 hours, respectively; Figure 8A) as demonstrated by
an increase in latency to step-down during training session and test sessions. However, when
the animals were pre-treatedwith scopolamine 1.0 µmol/Kg, neuroprotective the effect of
LQFM032 was reversed (Figura 8B).
3.10. Influence of nicotinic receptor on anxiolytic-like activity of LQFM032
The effect of LQFM032 on the number of transitions (Figure 9A) and time spent in
light area (Figure 9B) of LDB was blocked by mecamylamine.
4.0. Discussion
For preliminary pharmacological screening, gross behavior or Irwin test was carried
out. Present findings show that the treatment with LQFM032 induced behavioral alterations
such as sedation, ataxia, paralysis of the hindlimbs, that indicate central depressant activity.
This test is often used to evaluate the effects of a new compound on physiological and
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behavioral functions. Irwin test permits the determination of potential toxicity, appropriate
[23, 24,
dose and routes of administration.
25]. Moreover, always that possible test is
recommended, for detection potential adverse effects of candidate prototypes of drugs on the
central nervous system [26]. Based on these results, we choice to administer LQFM032 at 18-
162 µmol/Kg p.o. for the pharmacological tests.
Based effects on gross behavior in doses of 18 µmol/Kg, 54 µmol/Kg and 162
µmol/Kg were chosen for evaluation central pharmacological activity of LQFM032. The first
test to evaluate specific central activity of LQFM032 was sodium pentobarbital-induced sleep
test. This test permits observation of putative CNS depressant and/or stimulant activity
because of synergism or antagonism to the depressant activity of sodium pentobarbital [27, 28]
.
In the present study, the treatment with LQFM032 decreased sleep latency and increase sleep
time, thereby suggesting CNS depressant effect. This result is consistent with the previous
data on LQFM008 which also demonstrated CNS depressant activity [11]
.
Compounds with central depressant activity may compromise exploratory activity,
which could compromise the results in specific behavioral tests. Hence, open field test was
conducted to observe various behavioral parameters, such as rearings and numbers of
crossings that indicate exploration activity, grooming number indicating stereotyped activity,
among other behaviors including that may suggest an anxiolytic-like activity, which are
number of crossings and time spent in the centre of the open field[29, 30]. In the open field test
the treatment with LQFM032 (high dose) altered the capacity of locomotion, indicating
central depressant activity. Moreover, LQFM032, also at all doses tested, showed anxiolytic-
like activity by increased number of crossings and time spent at the center of the open field.
To exclude the possibility that LQFM032 may compromised capacity of motor
activity the wire hanging test was employed. In this test, the compound did not alter the fall
latency, indicating that the compound did not alter motor activity.
Altogether, the results of sodium pentobarbital-induced sleep test, open field test, and
wire hanging test showed the central depressant activity and suggested anxiolytic-like activity
of LQFM032. In order to evaluatant the anxiolytic-like activity of this compound, the EPM
and LDB tests were conducted. The EPM has been one of the most used behavioral model,
that consist of the analysis of the relative time spent in open arms expressed as the percentage
and ratio of the number of entries into these arms[31, 32, 33]. In the LDB, parameters such as the
number of transitions and the time spent in the light area were used to evaluate anti-anxiety
activity of test compound [33]. In these tests, LFQM032 demonstrated anxiolytic-like activity.
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In the EPM, the compound LQFM032 increased time spent and entries in the open arms, and
increased time spent in the light area and number of transitions in the LDB.
The anxiolytic-like activity of LQFM032 was reversed by flumazenil, benzodiazepine
site antagonist [34]. In contrary, NAN-190, 5-HT1A antagonist, did not block the effect of this
compound. Hence, these results suggest that the anxiolytic-like activity of LQFM032 is
benzodiazepine site dependent. These results are different from the previous data which
implicated 5-HT1A receptor in the anxiolytic-like activity of LQFM008 (analog of LQFM032)
[11]
.
Because the anxiolytic effects of classical agonists of benzodiazepines site are
associated with undesirable anterograde amnesia [35], it becomes necessary to evaluate the
effect of LFQM032 on the performance of experimental animal’s memory. In avoidance
memory in stepdown test, the treatment with LQFM032 increased the latency to stepdown
compared with test session, however the control group showed the same effect. This result
suggests that LQFM032 did not improve mmenomic activity in the same way the control
group (vehicle), so LQFM032 did not alter this effect.
On the other hand, LQFM032 did not protect the animals of the amnesic effect of
scopolamine, muscarinic agonist. Scopolamine produces a impairment in acquisition of new
knowledge. The amnesic action produced by scopolamine has been widely used as an
experimental model for evaluated drugs with potential cognitive enhancing abilitiy [36].
The LQFM032 showed anxiolytic-like activity without altering mnemonic activity
necessitated investigation of the involvement of nicotinic receptor in the anxiolytic-like
activity. The pretreatment with mecamylamine antagonist nicotinic [37, 38], reverted the effect
of LQFM032 in the LDB by decreasing number of transitions and time in the light area, when
compared with group treated with LQFM032.
LQFM032 did not produce a neuroprotective effect when the animals were pre-treated
with scopolamine.
The benzodiazepine site dependent effect of LQFM032 without mnemonic impairment
can be explained by activation of nicotine acetylcholine receptors that have been reported to
activate GABAergic activity. This result suggest that there is a direct relationship between the
GABAergic and nicotinic systems whereby acetylcholine activates nicotinic receptors on
GABA interneurons in the hippocampus. Whenever the cholinergic pathway is overactivated,
the first response is further activation of the GABA interneuron [38, 39]. Nicotinic receptor are
known to improve performance on attention and memory tasks [40]. Besides that, nicotinic
agonism showed anxiolytic-like activity [41]
.
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Nicotinic agonism effect of LQFM032 can be explained by prototype JWB-1-84-1
which in the structural design of compound LQFM032. JWB-1-84-1 is a choline analog whith
nicotinic agonism effect [42]. Previous reports has shown that JWB-1-84-1 is a tertiary amine
analogs of choline synthesized with expectation that they would be high potency compounds
for cytoprotection, this compound improved cognitive activity[13, 43]
.
Thereby, the hybridization molecular strategy from LQFM008 (2) and JWB-1-84-1 (3)
allowed to obtain a new piperazine derivative, LQFM032. This new compound showed
pharmacology central activity involved benzodiazepine site and nicotinic receptor. These
mechanism of action suggested a co-transmission involved nicotinci pathway and modulated
benzodiazepine site.
5.0. Conclusion
In summary, LQFM032 showed anxiolytic-like activity that suggest involvement of
benzodiazepine site and nicotinic receptors without altering mnemonic activity. Futures
studies will be focused on the investigation of possible co-transmission of acetylcholine and
GABA, as well as anti-depressant like activity of LQFM032.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors are grateful to FUNAPE/UFG, FAPEG, PROCAD/CAPES and CNPq for
financial support.
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Figure 1. (A) The structural design of LQFM032 (4) from LQFM 008 (2) and JWB-1-84-1
(3). (B) Synthetic route for the preparation of 2-(4-((1-phenyl-1H-pyrazol-4-
yl)methyl)piperazin-1-yl)ethanol (4) (LQFM032).
Figure 2: (A) Infrared spectrum of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-
1
yl)ethanol (4) – LQFM032. (B) H NMR expanded spectrum, aromatic region, of -(4-((1-
phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanol (4) – LQFM032 in CDCl3. (C) 13C
NMR spectrum of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanol (4) –
LQFM032 in CDCl3. (D) Mass spectrum of 2-(4-((1-phenyl-1H-pyrazol-4-
yl)methyl)piperazin-1-yl)ethanol (4) – LQFM032.
Figure 3. Effects of LQFM032, at different doses (p.o), and vehicle 10 mL/Kg (Tween 80
2%) in sodium pentobarbital-induced sleep test as evaluated in mice, (A) latency to (s.) and
(B) duration of sleep time (min.). Results are expressed as mean SEM; n = 8 each group.
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**p≤0.01, ***p≤0.001 – compared with the control group, one-way ANOVA followed by
Student-Newnman-Keuls’ test.
Figure 4. Effects of LQFM032 at doses of 18, 54 and 162 µmol/Kg or vehicle 10 mL/Kg
(Tween 80 2%) on the (A) percentage of entries into open arms, and (B) time spent in the
open arms. Results are expressed as mean SEM; n = 8. **p≤0.01, ***p≤0.001 – compared
with the control group, one-way ANOVA followed by Student-Newman-Keuls’ test.
Figure 5. Effects at differents doses of LQFM032 and control group (vehicle 10 mL/Kg –
Tween 80 2%) on (A) number of transitions between light and dark compartments, and (B)
time spent in the light area on LDB. Results are expressed as mean SEM; n = 8. **p≤0.01,
***p≤0.001 – compared with the control group, one-way ANOVA followed by Student-
Newnman-Keuls’ test.
Figure 6. (A) Effects of the pre-treatment with saline 0.9%, flumazenil 6.6 µmol/Kg or NAN-
190 1.3 µmol/Kg on the effects of LQFM032 54 µmol/Kg in the number of transitions
between compartments on LDB. (B) Effects of the pre-treatment with saline 0.9% or NAN-
190 1.3 µmol/Kg on the effects of buspirone 26 µmol/Kg in the number of transitions on
LDB. (C) Effects of the pre-treatment with saline 0.9% or flumazenil 6.6 µmol/Kg on the
effects of diazepam 3.51 µmol/Kg in the number of transitions on LDB. The same control
group (saline + vehicle) was for all figures. Results are expressed as mean SEM; n = 8.
*p≤0.05, ***p≤0.001 – compared with the control group, and ##p≤0.01, ###p≤ 0.001 –
compared with group treated with saline/LQFM032 (A), or saline/buspirone (B) or
saline/diazepam (C), one-way ANOVA followed by Student-Newnman-Keuls’ test.
Figure 7. (A) Effects of the pre-treatment with saline 0.9%, flumazenil 6.6 µmol/Kg or NAN-
190 1.3 µmol/Kg on the effects of LQFM032 54 µmol/Kg on time spent in light area (s) on
LDB. (B) Effects of the pre-treatment with saline 0.9% or NAN-190 1.3 µmol/Kg on the
effects of buspirone 26 µmol/Kg on time spent in light area (s) on LDB. (C) Effects of the
pre-treatment with saline 0.9% or flumazenil 6.6 µmol/Kg on the effects of diazepam 3.51
µmol/Kg p.o. on time spent in light area (s) on LDB. The same control group (saline +
vehicle) was for all figures. Results are expressed as mean
SEM; n = 8. *p≤0.05,
***p≤0.001 – compared with the control group, and ##p≤0.01, ###p≤ 0.001 – compared with
group treated with saline/LQFM032 (A), or saline/buspirone (B) or saline/diazepam (C), one-
way ANOVA followed by Student-Newnman-Keuls’ test.
Figure 8. (A) Effects at doses of LQFM032 and vehicle (control group – Tween 80 2%) on
memory evaluated in the step-down avoidance task, represented by latency to step-down (s).
(B) Effects of the pre-treatment with vehicle 10 mL/Kg and LQFM032 54 µmol/Kg on the
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effects of saline 0.9% or scopolamine 1.0 µmol/Kg in memory impairment in mice. TS –
training session. Results are expressed as mean SEM; n = 8. *p≤0.05, ***p≤0.001 –
compared to the training session of the respective group. One-way ANOVA followed by
Student-Newnman-Keuls’ test.
Figure 9. (A) Effects of the pre-treatment with saline 0.9% or mecamylamine 30 µmol/Kg on
the effects of LQFM032 54 µmol/Kg on number of transitions. (B) Effects of the pre-
treatment with saline 0.9% or mecamilamine 30 µmol/Kg on the effects of LQFM032 54
µmol/Kg on time spent in light area (s) of light-dark box. Results are expressed as mean
SEM; n = 8. **p≤0.01 – compared with the control group, and #p≤0.05; ###p≤0.001 –
comparison between the groups Mecamilamine+LQFM032 vs Saline+LQFM032.
Table 1. Effects of the LQFM032, at different doses, and vehicle 10 mL/Kg (Tween 80 2%)
in the open field and wire hanging tests.
LQFM032
Vehicle
18 µmol/Kg
54 µmol/Kg
162 µmol/Kg
10 ml/Kg
Open Field Test
Crossings
Rearings
Grooming
Imobility (s.)
%CrCe
114.7 1.92
44.29 2.85
1.00 0.38
1.78 0.36
27.88 1.43
72.67 1.80
111.7 2.71
43.43 2.82
1.11 0.31
1.80 0.33
36.63 2.04*
106.3 3.07
36.29 2.02*
0.57 0.30
1.67 0.33
49.14 2.63*** 42.29 3.04***
102.0 2.47**
33.71 1.54*
0.50 0.27
2.30 0.30
101.70 3.07*** 105.3 6.37*** 105.7 3.91***
TCe(s)
Wire Hanging Test
13.67 1.12
14.88 1.32 12.38 0.86 10.63 0.59
Fall latency (s.)
Results are expressed as mean SEM; n = 8 each group. *p≤0.05, **p≤0.01, ***p≤0.001 –
compared with the control group, one-way ANOVA followed by Student-Newnman-Keuls’
test. In the wire hanging test, the data are analyzed by the Kruskal-Wallis test followed by
Dunns as post hoc test and expressed as median (25th percentile to 75th percentile), n=8.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

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This article is protected by copyright. All rights reserved.

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