Anti-inflammatory effect of a new piperazine derivative: (4-methylpiperazin-1-yl)(1-phenyl-1H-pyrazol-4-yl)methanone

Article abstract of DOI:10.1007/s10787-017-0390-8

Aims: This study investigates the anti-nociceptive and anti-inflammatory effects of new piperazine compound (LQFM182) as well as the toxicity acute in vitro. Main methods: To evaluate the anti-nociceptive activity, the acetic acid-induced abdominal writhing test, tail flick test and formalin-induced pain test were used. The anti-inflammatory activity was evaluated using the models of paw oedema and pleurisy induced by carrageenan and some inflammatory parameters were evaluated, including cell migration, myeloperoxidase enzyme activity and the levels of TNF-α and IL-1β cytokines in pleural exudate. The acute oral systemic toxicity of LQFM182 in mice was evaluated through the neutral red uptake (nru) assay. Key findings: LQFM182 (50, 100 or 200?mg/kg, p.o.) decreased the number of writhings induced by acetic acid in a dose-dependent manner, and an intermediate dose (100?mg/kg, p.o.) reduced the paw licking time of animals in the second phase of the formalin test. Furthermore, LQFM182 (100?mg/kg, p.o.) reduced oedema formation at all hours of the paw oedema induced by carrageenan test and in pleurisy test reduced cell migration from the reduction of polymorphonuclear cells, myeloperoxidase enzyme activity and the levels of pro-inflammatory cytokines IL-1β and TNF-α. Therefore, it was classified in GHS category 300?50?

Full text of DOI:10.1007/s10787-017-0390-8

Inflammopharmacol
DOI 10.1007/s10787-017-0390-8
Inflammopharmacology
ORIGINAL ARTICLE
Anti-inflammatory effect of a new piperazine derivative:
(4-methylpiperazin-1-yl)(1-phenyl-1H-pyrazol-4-yl)methanone
1
2
2
2
•
Daniel C. Batista
Merita P. Gon c¸ alves
•
Daiany P. B. Silva
2
•
Iziara F. Florentino
3
•
Carina S. Cardoso
4
5
•
Marize C. Valadares
2
•
Luciano M. Li a˜ o
•
Germ a´ n Sanz
•
5
1
Boniek G. Vaz
•
Elson A. Costa
•
Ricardo Menegatti
Received: 5 July 2017 / Accepted: 12 August 2017
Ó Springer International Publishing AG 2017
Abstract
Aims This study investigates the anti-nociceptive and anti-
inflammatory effects of new piperazine compound
in a dose-dependent manner, and an intermediate dose
(100 mg/kg, p.o.) reduced the paw licking time of animals
in the second phase of the formalin test. Furthermore,
LQFM182 (100 mg/kg, p.o.) reduced oedema formation at
all hours of the paw oedema induced by carrageenan test
and in pleurisy test reduced cell migration from the
reduction of polymorphonuclear cells, myeloperoxidase
enzyme activity and the levels of pro-inflammatory
cytokines IL-1b and TNF-a. Therefore, it was classified in
GHS category 300 \ LD \ 2000 mg/kg.
(
LQFM182) as well as the toxicity acute in vitro.
Main methods To evaluate the anti-nociceptive activity,
the acetic acid-induced abdominal writhing test, tail flick
test and formalin-induced pain test were used. The anti-
inflammatory activity was evaluated using the models of
paw oedema and pleurisy induced by carrageenan and
some inflammatory parameters were evaluated, including
cell migration, myeloperoxidase enzyme activity and the
levels of TNF-a and IL-1b cytokines in pleural exudate.
The acute oral systemic toxicity of LQFM182 in mice was
evaluated through the neutral red uptake (nru) assay.
Key findings LQFM182 (50, 100 or 200 mg/kg, p.o.)
decreased the number of writhings induced by acetic acid
5
0
Significance Reduction of the TNF-a and IL-1b levels.
Keywords Anti-inflammatory Á N-arylheterocycles Á
IL-1b Á TNF-a
Introduction
Electronic supplementary material The online version of this
article (doi:10.1007/s10787-017-0390-8) contains supplementary
material, which is available to authorized users.
The inflammatory process is defined as a protective,
dynamic and adaptive reaction of the human body against
any aggressive agent (Nathan and Ding 2010; Ashley et al.
&
Ricardo Menegatti
rm_rj@yahoo.com
2012). This process is characterised by the production and
release of pro-inflammatory mediators, such as histamine,
bradykinin, prostaglandins, leukotrienes and cytokines,
which leads to the appearance of cardinal signs of
inflammation including pain, heat, redness and tumour; if
there is no proper resolution of the inflammatory process, it
may result in the loss of function of the affected tissue
(Nathan 2002; Medzhitov 2008).
1
Faculty of Pharmacy, Laboratory of Medicinal
Pharmaceutical Chemistry, Federal University of Goi a´ s,
Goi aˆ nia, GO, Brazil
2
Department of Pharmacology, ICB, Federal University of
Goi a´ s, Campus Samambaia, 314, Goi aˆ nia, GO 74001-970,
Brazil
3
4
5
Laboratory of Pharmacology and Cell Toxicology, Faculty of
Pharmacy, Federal University of Goi a´ s, Goi aˆ nia, GO, Brazil
Among the pro-inflammatory mediators is the histamine
which belongs to class of vasoactive amines. Histamine is
produced and stored primarily in mast cells and basophils
from the decarboxylation of L-acid–amine histidine and
performs its physiological functions by stimulating his-
taminergic receptors, which are well-known as H R, H R,
Chemistry Institute, Federal University of Goias, Campus
Samambaia, Goi aˆ nia, GO, Brazil
Chemistry Institute, Laboratory of Chromatography and Mass
Spectrometry-LaCEM, Federal University of Goi a´ s, Goi aˆ nia,
GO, Brazil
1
2
123

D. C. Batista et al.
H R and H R (Parsons and Ganellin 2006; Kiss et al.
3
pyrazole-4-carboxylic acid (6) (188.06 mg, 1 mmol),
SOCl (4 mL, 55.14 mmol) and a few drops of anhydrous
4
2
008). The H4 receptor, expressed in the membrane of
2
immune cells, is responsible for histamine-induced
chemotaxis and, therefore, is a potential target for the
design and synthesis of new compounds for treating
chronic inflammatory diseases (Zampeli and Tiligada
DMF were added to make one solution. The mixture was
heated at 80 °C for 3 h. In turn, the excess SOCl was then
2
evaporated and N-methylpiperazine (7) (0.14 mL, 1 mmol),
triethylamine (0.17 mL, 1 mmol) and CH Cl (4 mL) were
2
2
2
009; Kiss and Keseru 2014). Moreover, there are reports
added to the residue; the new mixture was kept at room
temperature for 2 h. At the end of the reaction, the residue
was split between water and CH Cl . The phases were
of the involvement of H receptors in the differentiation
4
and metastasis of various neoplasic diseases such as breast
2
2
(
Medina et al. 2008; Tiligada et al. 2009) and lung cancer
Cai et al. 2014), eye diseases such as macular degenera-
separated and the aqueous layer was extracted into
(
3 9 15 mL with CH Cl and the combined organic layers
2
2
tion (Kaneko et al. 2014), vestibular disorders (Wersinger
et al. 2013) and ulcerogenesis (Adami and Coruzzi 2014;
Coruzzi et al. 2011).
were dried (Na SO ), concentrated in vacuo, and the crude
2 4
product was purified by column chromatography (SiO2,
CH Cl /MeOH = 98:2) to (4-methylpiperazin-1-yl)(1-
2
2
Prototypes (JNJ7777120, VUF6002 and VUF6007) have
recently been developed which act to antagonise the H4
receptor, exerting anti-inflammatory and anti-nociceptive
activities (Thurmond et al. 2004a, b; Coruzzi et al. 2007;
Kiss and Keseru 2014). In the future, these compounds and
new analogues could be used as therapeutic as well as
pharmacological tools.
phenyl-1H-pyrazol-4-yl)methanone (2) (190 mg, 70%) as a
beige solid, m.p. = 97–106 °C, Rf = 0,42 (CH Cl /
2
2
-
1
MeOH = 95:5). IRmax (KBr) cm : 3098 (m C–H), 2990 (m
1
C–H), 1606 (m C=O) and 1555 (m C=C); H-NMR
(500.13 MHz) CDCl (d): 8.23 (1H, d, J = 0.45 Hz; H-5);
3
7.82 (1H, d, J = 0.40 Hz, H-3), 7.68 (1H, dddd, J = 7.5;
0
2.2; 1.6 and 0.7 Hz; H-2 ); 7.68 (1H, dddd, J = 7.2; 2.2; 1.2
0
In the scope of a research programme aimed at devel-
oping new drugs for the treatment of central inflammatory
diseases, we describe the synthesis and biological evalua-
tion of LQFM182 (2), a new piperazine compound.
LQFM182 (2) was designed through a ring bioisosterism
strategy from the JNJ-7777120 (1) lead compound, a his-
tamine H4 antagonist (Fig. 1). We replaced the indole ring
present in JNJ-7777120 (1) with the 1-(phenyl)-1H-pyra-
zole scaffold present in LQFM182 (2), a strategy which has
been used with success in drug discovery programmes
and 0.9 Hz; H-6 ); 7.46 (1H, dddd, J = 7.5; 7.4; 1.9 and
0
0.9 Hz; H-3 ); 7.46 (1H, dddd, J = 7.4; 7.2; 1.9 and 0.7 Hz;
0
0
H-5 ); 7.33 (1H, tdd, J = 7.4; 1.6 and 1.2 Hz; H-4 ); 3.82
00
0
0
(4H, t, J = 4.55 Hz; H-2 and 6 ); 2.53 (4H, t,
0
0
00
00
J = 3.70 Hz; H-3 and 5 ); 2.38 (3H, s, H-4 ); 2D NMR
(HSQC/HMBC-125.76 MHz) CDCl /TMS (d): 162.9 (C-
3
0
0
0
0
6); 139.9 (C-1 ); 139.8 (C-3); 129.5 (C-3 and 5 ); 128.8 (C-
0
0
00
5); 126.9 (C-4 ); 119.4 (C-2 and 6 ); 118.8 (C-4); 54.8 (C-3
?
0
0
00
00
00
and 5 ); 45.9 (C-2 and 6 ); 45.5 (C-4 ); MS: [M?H] m/z
of 271.1551; purity[98% (Supporting Information)
(
Lima and Barreiro 2005).
Pharmacology
Materials and methods
Chemistry
Animals
Experiments were performed using female Swiss albino
mice (25–30 g) from the Central Animal House of the
Federal University of Goi a´ s (UFG). Animals were kept in
plastic cages at 22 ± 2 °C, with free access to pellet food
and water and under a 12-h light/dark cycle, in compliance
with the International Guiding Principles for Biomedical
Research Involving Animals. The animals were acclima-
tised for 7 days before the beginning of the experiments.
All experimental protocols were developed and approved
according to the principles of ethics and animal welfare
designated by the Ethics Committee on Animal Use of
UFG (Number: 017/13).
Synthesis of (4-methylpiperazin-1-yl)(1-phenyl-1H-pyra-
zol-4-yl)methanone—LQFM182 (2) (Ermondi et al. 1998).
To a heated heterogeneous mixture of 1-phenyl-1H-
Drugs and chemicals
The chemicals used in this study were acetic acid (Merck,
USA), DMSO (Sigma Chemical, USA), carrageenan
Fig. 1 Structural design of LQFM182 (2) from JNJ-7777120 (1)
1
23

Anti-inflammatory effect of a new piperazine derivative: (4-methylpiperazin-1-yl)(1-phenyl-…
Ò
(
Sigma Chemical, USA), dexamethasone (Decadron ,
mirror was placed under this box to enable the unhindered
observation of the formalin-injected paw for 30 min. Pain
reaction time (licking time) was assessed during two
periods: from 0 to 5 min, the first phase, where neurogenic
pain is caused by direct stimulation of the nociceptors, and
from 15 to 30 min, the second phase, where inflammatory
pain is caused by the release of inflammatory mediators.
These results were expressed as the mean ± SEM of
licking time in seconds.
Ache, Brazil), formaldehyde (Synth, Brazil), indomethacin
Ò
(
Indocid , Merck Sharp & Dohme Farmac eˆ utica-Ltda, SP,
Ò
Brazil), and morphine hydrochloride (Dimorf , Cristalia,
SP, Brazil). LQFM182 (2) was dissolved in 10% DMSO in
distilled water, and all other drugs were dissolved in dis-
tilled water. The doses of the LQFM182 (2) were based on
the doses of JNJ7777120 (1) used in previous studies
(
Hsieh et al. 2010).
Anti-nociceptive activity
Anti-inflammatory activity
Acetic acid-induced abdominal writhing test The acetic
acid-induced nociception was achieved as described pre-
viously by Koster et al. (1959). Groups of mice (n = 8)
were treated by gavage (p.o.) with vehicle (10% DMSO
Carrageenan-induced oedema Carrageenan-induced hind
paw oedema in mice was used as the animal model of acute
inflammation according to the method of Winter et al.
(1962). Initially, the animals (n = 8) were treated with
vehicle (10% DMSO, 10 mL/kg, p.o.), LQFM182 (2)
(100 mg/kg, p.o.) or indomethacin (10 mg/kg p.o.—posi-
tive control). One hour later, 50 lL of carrageenan (1%)
was injected intraplantar into the right hind paw and 50 lL
of saline (0.9% NaCl) into the left hind paw (used as the
control). The paw volume was measured using a plethys-
mometer (Model 7141, Ugo Basile, Italy) at 1, 2, 3 and 4 h
after the injection of carrageenan. The baseline was per-
formed before the treatments (time 0) for each animal. The
results were expressed in lL as mean ± SEM.
1
0 mL/kg), LQFM182 (2) at doses 50, 100 or 200 mg/kg
or indomethacin (10 mg/kg, positive control for anti-no-
ciceptive activity) 60 min before the application of acetic
acid solution (1.2% v/v; 10 mL/kg, i.p.). The number of
abdominal constrictions (writhing) was counted for each
animal, over a period of 30 min after acetic acid injection;
the results are expressed as the mean ± SEM of the
number of writhings.
Tail flick test The tail flick test was performed as
described previously by D‘Amour et al. (1941). In this test,
the time taken to flick the tail (latency) when it was
exposed to a heat source was measured using an analges-
Carrageenan-induced pleurisy The animals (n = 8) were
treated with vehicle (10% DMSO, 10 mL/kg, p.o.),
LQFM182 (2) (100 mg/kg, p.o.) or dexamethasone (2 mg/
kg, p.o.—positive control). One hour after the treatments,
the animals received an injection of 100 lL of 1% car-
rageenan into the pleural cavity. Four hours later, the
pleural exudate was collected with 1 mL of heparinised
phosphate-buffered saline and used to count the number of
total leukocytes using T u¨ rk solution in a Neubauer cham-
ber (Vinegar et al. 1973), to evaluate the myeloperoxidase
activity, the levels of TNF-a and IL-1b.
Ò
imeter (Insight , Ribeir a˜ o Preto, S a˜ o Paulo, Brazil). The
animals were divided into three experimental groups
(
(
n = 8): vehicle (10% DMSO, 10 mL/kg, p.o.), LQFM182
2) (100 mg/kg, p.o.) or morphine (5 mg/kg, s.c.—positive
control for anti-nociceptive activity). The latency baseline
was performed 30 min before the treatments (time -30)
and immediately after of the treatments (time 0) for each
animal. After, the latency to pain reaction was measured at
3
0, 60, 90, 120 and 150 min. A cut-off of 15 s was set to
prevent the risk of burns. The results were expressed as
mean ± SEM, in seconds, at the different times.
Activity of myeloperoxidase enzyme To measure the
myeloperoxidase (MPO) activity, 40 lL of pleural lavage
of mice treated with vehicle (10% DMSO, 10 mL/kg, p.o.),
LQFM182 (2) (100 mg/kg, p.o.) or dexamethasone (2 mg/
kg, p.o.—positive control) was transferred to eppendorfs.
The reaction was started by adding 360 lL of phosphate
buffer pH 6.0 containing 0.167 mg/mL of o-dianisidine 2
HCl and 0.0005% H O . The enzyme reaction was stopped
Formalin test Formalin-induced nociception was per-
formed as described previously by Hunskaar and Hole
(
1987). Groups of mice (n = 7) were treated with vehicle
10% DMSO 10 mL/kg p.o.), LQFM182 (2) (100 mg/kg
(
p.o.), indomethacin (10 mg/kg, p.o.—positive control for
anti-nociceptive activity in the second phase), or morphine
2
2
(
5 mg/kg, s.c.—positive control for anti-nociceptive
after 15 min by adding 30 lL of sodium azide 1%. The
samples were centrifuged subsequently for 5 min at
1000 rpm. The supernatant was separated and 200 lL was
transferred to microplate wells; absorbance was assayed by
enzyme-linked immunosorbent assay (ELISA), at a wave-
length of 450 nm (Sedgwick 1995; Saleh et al. 1999). The
activity in the first and second phases). Sixty min after the
p.o. treatment, or 30 min after s.c. treatment, 20 lL of 3%
formalin (in saline) was administered into the plantar sur-
face of the right hind paw. After the phlogistic agent
injection, the mice were placed into an acrylic box, and a
123

D. C. Batista et al.
results were expressed as means ± SEM of enzymatic
(IC ) ? 0.757. The estimated LD value obtained was
5
0
50
activity in mU/mL.
used to classify the probable acute oral systemic toxicity of
LQFM182 (2) according to the Globally Harmonised
Classification and Labelling of Chemicals (GSH) (OECD
2001).
Assay of TNF-a and IL-1b levels The pleural exudates of
mice treated with vehicle (10% DMSO, 10 mL/kg, p.o.),
LQFM182 (2) (100 mg/kg, p.o.) or dexamethasone (2 mg/
kg, p.o.—positive control) were also used to determine the
concentrations of TNF-a and IL-1b using an immunosor-
bent assay kit (ELISA) (Ebioscience) (Nicoletti et al. 2010;
Costa et al. 2013). The results were expressed as mean-
s ± SEM in pg/mL.
Statistical analysis
The data were analysed statistically by a one-way ANOVA
followed by the Newman–Keuls’ test as a post hoc or a
two-way ANOVA followed by the Bonferroni’s test as a
post hoc (Sokal and Rohlf 1981). All statistical analysis
was carried out using GraphPad Prism version 5.00. Values
of P B 0.05 were considered significant.
Toxicology
In vitro assay to estimate LD50 of LQFM182 (2)
Neutral red uptake (NRU) assay was carried out using
Balb/c 3T3 fibroblasts, based on the protocol described by
Borenfreund and Puerner (1985) modified by NICEATM
Results
Synthesis of (4-methylpiperazin-1-yl)(1-phenyl-1 h-
pyrazol-4-yl)methanone—LQFM182 (2)
(
ICCVAM 2006). In brief, the cells were cultured in
DMEM, supplemented with 10% (v/v) FBS, 100 IU/mL
penicillin, 100 lg/mL streptomycin and routinely grown as
a monolayer in tissue culture flasks (75 cm ) under stan-
The synthetic route (Fig. 2) began with a condensation
reaction between 1-(phenyl)hydrazine hydrochloride (3)
and 1,1,3,3-tetramethoxy propane through the work
described by Finar and Hurlock (Finar and Hurlock 1957)
to provide 1-(phenyl)-1-H-pyrazole (4) 88% of yields. The
chemoselective and regiospecific formylation of 1-(phe-
nyl)-1-H-pyrazole (4) to 1-(phenyl)-1-H-pyrazole-4-
carbaldehyde (5) was carried out under Duff’s conditions,
with an 83% yield (De Oliveira et al. 2013). In turn, the
1-(phenyl)-1-H-pyrazole-4-carbaldehyde (5) was oxidised
to compound 1-(phenyl)-1-H-pyrazole-4-carboxylic acid
2
dard culture conditions at 37 °C and 5% CO in a humid
2
environment. When the cells exceeded 70–80% conflu-
ence, they were removed from the culture flasks using
trypsinisation (trypsin/EDTA solution, 0.025:0.02%). Cell
viability was analysed using a TC20TM automated cell
counter (Hercules, CA, USA), according to the manufac-
turer’s instructions. A value of [90% was considered
acceptable for conducting the assays.
5
Thus, 3T3 fibroblasts (0.5 9 10 cells/mL) were seeded
in 96-well plates overnight. The cells were then treated
with vehicle (DMSO 0.6%) or nine different concentrations
of LQFM182 (2) (0.02–2.26 mM) in complete medium.
The blank wells (with no cells) received complete culture
medium with or without the test compounds. After 48 h,
the supernatant was discarded and 100 lL of NR (0.25 mg/
mL) diluted in DMEM containing SFB (5%, v/v) was
added to all wells, followed by incubation. After 3 h, the
NR medium was removed, the cells were washed with 100
lL/well of pre-warmed PBS and 100 lL of NR desorb (50
ethanol: 1 acetic acid: 49 ultrapure water) solution was
added to all wells. The plates were shaken on a microplate
shaker (Orbit P4, Labnet International, Edison, NJ, USA)
for 20 min and 35 rpm. Absorbance was measured at
(6) through the use of KMnO in water, with a 98% yield
4
(Shriner and Kleider 1943). Finally, the 1-(phenyl)-1-H-
pyrazole-4-carboxylic acid (6) reacted with SOCl , using
2
DMF as a catalyser to offer the acyl chloride intermediate
which, in turn, reacted with 1-methylpiperazine to provide
(4-methylpiperazin-1-yl)(1-phenyl-1H-pyrazol-4-yl)metha-
none—LQFM182 (2) at a yield of 70% (Ermondi et al.
1998). As we can see, LQFM182 (2) was obtained after
four synthetic steps, with a global yield of 50%.
Anti-nociceptive activity
Acetic acid-induced abdominal writhing test
5
50 nm in a spectrophotometer (Multiskan Spectrum,
In this test, the oral treatments with LQFM182 (2) at doses
50, 100 or 200 mg/kg decreased the number of writhings
induced by acetic acid in a dose-dependent manner com-
pared to the control group (vehicle 10% DMSO 10 mL/kg)
from 107.1 ± 6.8 (group treated with vehicle) to
85.7 ± 11 (20% less, P \ 0.05), 48.8 ± 3.3 (54.4% less,
P \ 0.001) and 45.6 ± 5.4 (57.4% less, P \ 0.001),
Thermo Scientific, Waltham, MA, USA). A curve was
obtained from the viability data for each concentration
LQFM182 (2). The concentration that inhibits cell growth
by 50% (IC ) was calculated. The lethal dose (LD ) was
5
0
50
estimated from the previously established in house model
equation (Vieira et al. 2011): Log (LD ) = 0.545 9 log
5
0
1
23

Anti-inflammatory effect of a new piperazine derivative: (4-methylpiperazin-1-yl)(1-phenyl-…
Fig. 2 Synthetic route to preparation of (4-methylpiperazin-1-yl)(1-phenyl-1H-pyrazol-4-yl)methanone (2) (LQFM182)
1
5
1
1
50
00
Vehicle 10 mL/kg p.o.
Vehicle 10 mL/kg p.o.
*
** ***
LQFM182 100 mg/kg p.o.
Morphine 5 mg/kg s.c.
LQFM182 50 mg/kg p.o.
LQFM182 100 mg/ p.o.
LQFM182 200 mg/kg p.o.
Indomethacin 10 mg/kg p.o.
*
**
10
5
*
*
**
***
*
**
*
**
***
5
0
0
0
-
30
0
30
60
90
120 150
Fig. 3 Effect of LQFM182 (2) (50, 100 and 200 mg/kg p.o.) on the
number of acetic acid-induced writhings in mice (n = 8). Indometha-
cin (10 mg/kg p.o.) was used as a positive control. Vertical bars
represent mean ± SEM of number of writhings in 30 min for each
experimental group. *P B 0.05 and ***P B 0.001 (compared with
control group) according to ANOVA followed by post hoc Newman–
Keuls’ test. Indo indomethacin
Time (min.)
Fig. 4 Effect of LQFM182 (2) on the tail-flick test in mice (n = 8).
The oral treatment with LQFM182 (2) (100 mg/kg p.o.) did not
change latency to thermal stimulation compared to the control group.
Morphine (5 mg/kg s.c.) was used as a positive control. The values
are expressed as mean ± SEM of the latency for the nociceptive
behaviour, in seconds. ***P B 0.001 (compared with control group)
according to two-way ANOVA followed by Bonferroni’s post hoc
test
respectively. The positive control for this test, indometha-
cin (10 mg/kg), reduced the number of writhings by 48%
(
55.43 ± 5.7, P \ 0.001) (Fig. 3).
Tail flick test
of 72.5 ± 3.3 (vehicle group) to 7.29 ± 2.8 (90% less,
P \ 0.001) and the second phase to 8.09 ± 0.8 (a reduc-
tion of 95%, P \ 0.001).
In the thermal nociception tail-flick test, treatment with
LQFM182 (2) (100 mg/kg, p.o.) manifested no significant
anti-nociceptive activity compared to the control group
Anti-inflammatory activity
(
vehicle). Morphine (5 mg/kg, s.c.—opioid agonist)
demonstrated significant anti-nociception at the times of
0, 60, 90, 120 and 150 min after treatment (Fig. 4).
Carrageenan-induced oedema
3
The oral treatment with LQFM182 (2) at a dose of 100 mg/
kg reduced paw oedema in all hours of the test. In the first
hour, LQFM182 (2) reduced the difference between the
paws from 142.5 ± 5.6 to 115.7 ± 6.8 lL (19% reduction,
P \ 0.01); in the second hour from 140 ± 6.3 to 88.6 ± 7
lL (37% reduction, P \ 0.001); in the third hour from
130 ± 3.8 to 85.7 ± 6.1 lL (34% reduction, P \ 0.001);
and finally, in the fourth hour from 126.7 ± 6.9 to
77.1 ± 5.2 lL (39% reduction, P \ 0.001). The treatment
with indomethacin (10 mg/kg, p.o.) reduced the paw
oedema in all hours of the test to 92.4 ± 4.6 (35%
reduction, P \ 0.001), 77.2 ± 4.4 lL (45% reduction,
P \ 0.001), 61.3 ± 3.8 (53% reduction, P \ 0.001) or
61 ± 7.5 lL (52% reduction, P \ 0.001), respectively,
compared to vehicle (Fig. 6).
Formalin test
In the formalin test, LQFM182 (2) in an intermediate dose
(
100 mg/kg, p.o.) showed anti-nociceptive activity com-
pared to the control group only in the second phase of the
test (Fig. 5). In the second phase, the vehicle group showed
a licking time (s) of 179.3 ± 10.7; LQFM182 (2) reduced
this time to 68.3 ± 14.6 (62% less, P \ 0.001). The group
treated with indomethacin (10 mg/kg, p.o.), the anti-in-
flammatory positive control, also showed a decreased
licking time (s) in the second phase, to 111.2 ± 7.2 (a
reduction of 38%, P \ 0.001), as expected. The positive
control morphine (5 mg/kg, s.c.) decreased the first phase
123

D. C. Batista et al.
1st Phase
2nd Phase
1
00
200
Vehicle 10 mL/kg p.o.
LQFM182 100 mg/kg p.o.
Indomethacin 10 mg/kg p.o.
Morphine 5 mg/kg s.c.
8
6
4
2
0
0
0
0
0
1
1
50
00
***
***
5
0
0
*
**
***
Fig. 5 The anti-nociceptive effect of LQFM182 (2) on the pain
induced by the formalin test in mice (n = 8). Oral treatment with
LQFM182 (2) (100 mg/kg) reduced the licking time in only the
second phase of the test. Indomethacin (10 mg/kg, p.o.) and morphine
(0–5 min) and second phase (15–30 min). Vertical bars represent
mean ± SEM of licking time of paw, in seconds. ***P B 0.001
compared with control group, according to ANOVA followed by post
hoc Newman–Keuls’ test
(
5 mg/kg, s.c.) were used as positive controls, during the first
6
0.5 ± 6.4
(43%
reduction,
P \ 0.001)
or
1
1
60
20
Vehicle 10 mL/kg p.o.
29.8 ± 15.6 mU/mL (72% reduction, P \ 0.001), respec-
tively, compared to the vehicle-treated group (enzymatic
activity 106.7 ± 18 mU/mL) (Fig. 7b).
LQFM182 100 mg/kg p.o.
Indomethacin 10 mg/kg p.o.
**
**
*
**
*
**
*
***
**
8
0
0
0
***
*
*
**
Assay of TNF-a and IL-1b levels The levels of TNF-a
was reduced by oral treatment with LQFM182 (2)
(100 mg/kg) or with dexamethasone (2 mg/kg) to
4
0
1
2
3
4
2.6 ± 0.5 (87% reduction, P \ 0.001) or 3.5 ± 1.8 (83%
Time (Hours)
reduction, P \ 0.05), respectively, compared to the vehicle
treated group (TNF-a: 20.2 ± 4.1 pg/mL) (Fig. 7c). As
can be observed in Fig. 7d, these some treatments signifi-
cantly reduced the levels of IL-1b interleukin to
Fig. 6 Effect of LQFM182 (2) on the carrageenan-induced oedema
test in mice (n = 8). The oral treatment with LQFM182 (2) at
1
(
00 mg/kg reduced the oedema at all hours of the test. Indomethacin
10 mg/kg, p.o.) was used as positive control. The values were
expressed as mean ± SEM of the difference between the paws, in lL.
P B 0.05, **P B 0.01 or ***P B 0.001, compared to control group,
428.3 ± 17.4
(26%
reduction,
P \ 0.001)
or
*
85.2 ± 9.7 pg/mL (85.3% reduction, P \ 0.001), respec-
tively, compared to the vehicle group (IL-1b:
according to two-way ANOVA followed by Bonferroni’s post hoc
test
579 ± 13.3 pg/mL).
In vitro assay to estimate LD50 value
Carrageenan-induced pleurisy
The 3T3 cells were exposed to different concentrations of
LQFM 182 (2) (0.02–2.26 mM) for 48 h and cell viability
was assessed by the NRU assay. This compound promoted
cell death in a concentration-dependent manner. The value
Cell migration In the test of carrageenan-induced
pleurisy, the oral treatments with LQFM182 (2) (100 mg/
kg) or dexamethasone (2 mg/kg) reduced the total number
of leukocytes to 4.06 ± 0.43 (34.5% reduction, P \ 0.001)
6
or 2.0 ± 0.56 leukocytes 9 10 /mL (68% reduction,
of IC50 obtained was 0.832 mM (Fig. 8). From this IC50
value, the LD50 was estimated, using previously estab-
P \ 0.001), respectively, compared to the control group
(
6
6.2 ± 0.77 leukocytes 9 10 /mL). In the differential
lished in house model equation (Vieira et al. 2011), to be
1
397 mg/kg. Thus, this compound was classified according
count of leukocytes, it was observed that the oral treatment
with LQFM182 (2) at dose 100 mg/kg significantly
reduced the migration of the polymorphonuclear leuko-
cytes from 5.2 ± 0.34 (vehicle group) to 3.3 ± 0.43
to GHS as category 4, LD [ 300 \ 2000 mg/kg.
50
Discussion
(
36.5% reduction, P \ 0.001). The positive control dex-
amethasone also significantly reduced the migration of the
polymorphonuclear leukocytes to 0.61 ± 0.2 (88% reduc-
tion, P \ 0.001) (Fig. 7a).
The present study reported the synthesis and preliminary
pharmacological profile of a new heterocyclic compound
(2) designed through ring the bioisosterism strategy from
JNJ-7777120 (1), the lead compound.
Activity of myeloperoxidase enzyme The oral treatment
with LQFM182 (2) (100 mg/kg) or dexamethasone (2 mg/
kg) also reduced the activity of myeloperoxidase enzyme to
In order to evaluate the possible anti-nociceptive and/or
anti-inflammatory activity of LQFM182 (2), the acetic
1
23

Anti-inflammatory effect of a new piperazine derivative: (4-methylpiperazin-1-yl)(1-phenyl-…
Fig. 7 Effect of LQFM182 (2)
in carrageenan-induced pleurisy
in mice (n = 8). The figure
shows the effect of LQFM182
A
B
8
150
6
(
2) (100 mg/kg, p.o.) or
dexamethasone (2 mg/kg p.o.)
on a cell migration,
100
50
0
*
4
2
0
*
*
b myeloperoxidase activity,
c TNF-a levels and d IL-1b
levels. The bars represent the
mean ± SEM of number of
migrated leukocytes/mL 9 106
to pleural cavity, the
*
**
***
*
**
TotalPoli
Mono
mean ± SEM of enzymatic
activity MPO in mU/mL and the
mean ± SEM of TNF-a and IL-
C
D
3
2
0
800
1
b levels in pg/mL in the
pleural exudate. *P \ 0.05,
*P \ 0.01 and ***P \ 0.001,
6
4
2
00
00
00
0
0
*
***
compared to control group,
according to ANOVA followed
by Newman–Keuls’ test
10
0
*
*
*
**
*
*
Vehicle 10 mL/kg p.o.
LQFM182 100 mg/kg p.o.
Dexamethasone 2 mg/kg p.o.
number of writhing induced by acetic acid suggesting a
nociceptive activity for the new compound. Similar results
have been described with other piperazine derivatives,
which have showed potential analgesic effect for the
treatment of pain (Chen et al. 2011a, b; Chae et al. 2012).
In the next tests, only the intermediate dose of LQFM182
(2) was used to reduce the number of animals as recom-
mended by the CEUA.
To investigate the possible involvement of central
mechanism in the anti-nociceptive effect of LQFM182 (2),
the tail-flick was conducted. This test uses the thermal
stimulus to evaluate an anti-nociceptive activity mediated
by spinal mechanism (D’amour and Smith 1941; Barrot
2012). LQFM182 (2) at dose used (100 mg/kg, p.o.) did
not increase the latency to thermal stimulus in either test,
suggesting that the anti-nociceptive effect of new com-
pound in this study is due to peripheral mechanisms.
In addition, this likely peripheral effect was confirmed
in the formalin test that identified two distinct phases of
pain. The first phase is characterised by the direct stimu-
lation of nociceptive fibres by formalin action and the
release of preformed mediators such as serotonin, kinins
and histamine, whereas the second phase is associated with
the formation and release of inflammatory mediators such
as cytokines and eicosanoids (Hunskaar and Hole 1987;
Shibata et al. 1989; Rosland et al. 1990; Tjolsen et al.
1992). It was observed that LQFM182 (2) (100 mg/kg)
showed anti-nociceptive activity only in the second phase
of the test. Shibata et al. (1989) have shown that central
Fig. 8 Cytotoxicity evaluation of LQFM182 (2) (0.02–2.26 mM) in
T3 cells by neutral red uptake (NRU) assay. The 3T3 fibroblasts
(
cells were then treated with vehicle (DMSO 0.6%) or LQFM182 (2)
in complete medium. After 48 h of exposure, the cell viability was
evaluated using neutral red dye
3
5
0.5 9 10 cells/mL) were seeded in 96-well plates overnight. The
acid-induced abdominal writhing test was performed. The
administration of acetic acid in the peritoneal cavity
induces acute inflammatory processes from the action of
acid and the liberation of chemical mediators such as
bradykinin, substance P, prostaglandins and cytokines
(
Ribeiro et al. 2000; Zakaria et al. 2006; Malvar et al.
014). After the application of acetic acid, abdominal
2
contraction and extension was observed, followed by one
or both hind legs of the mice being rotated. In this test, the
oral treatments with LQFM182 (2) at doses 50, 100 or
2
00 mg/kg decreased in a dose-dependent manner the
123

D. C. Batista et al.
acting analgesics such as narcotics can reduce both phases
of test, whereas anti-inflammatory drugs primarily suppress
the late phase of nociception. Therefore, ours results sug-
gest that LQFM182 (2) has anti-inflammatory activity.
Similar result has been seen with non-steroidal anti-in-
flammatory drugs (Ortiz 2012; Zhao et al. 2017).
to evaluate the real association of LQFM182 (2) and the
expression of NF-kappa b to better understand the mech-
anisms involved in the anti-inflammatory effect of this
compound.
In addition, in the attempt to evaluate the toxicity of
LQFM182 (2), an in vitro assay was performed to estimate
the oral acute LD50 value of this compound. The value
found was 1397 mg/kg, which allowed classified the
compound according to GHS as category 4. This category
includes substances with a low oral acute toxicity.
Previous research has reported an important role of
piperazine derivatives in the control of pain and inflam-
mation in different experimental models in vivo, showing a
reduction of pain, oedema and pro-inflammatory cytokines
(
Rordorf-Adam et al. 1994; Thurmond et al. 2004a, b;
Jiang et al. 2008; Chen et al. 2011a, b; Silva et al. 2015).
To confirm the anti-inflammatory effect of LQFM182
Conclusion
(
2), the carrageenan-induced paw oedema test was per-
formed first. Carrageenan promotes the development of an
anti-inflammatory process with the consequent formation
of oedema by the action of multiple inflammatory media-
tors, such as histamine and prostaglandins (Winter et al.
We can conclude that LQFM182 (2) showed satisfactory
anti-inflammatory activity which has been shown to cor-
relate with a decrease in TNF-a levels. In addition, in acute
toxicity assays, the prototype was shown to be safe for oral
administration.
1
962; Di Rosa et al. 1971; Liew and McInnes 2002; Morris
003). The results show that the treatment of animals with
2
Acknowledgements The authors are grateful to FUNAPE/UFG,
PROCAD/CAPES and CNPq for financial support.
LQFM182 (2) at a dose of 100 mg/kg reduced the oedema
formation in all hours of the test, confirming the anti-in-
flammatory effect observed in the formalin test.
Compliance with ethical standards
In addition, the carrageenan-induced pleurisy test was
performed. In this test, it is possible to evaluate and
quantitate several inflammatory parameters, such as cell
migration, myeloperoxidase activity and cytokine levels
from the pleural exudate collected (Vinegar et al. 1973;
Higgs et al. 1980; Mikami and Miyasaka 1983). From the
analysis of the pleural exudate, the results showed that
LQFM182 (2) reduced the cell migration from the reduc-
tion of polymorphonuclear cell migration. Moreover,
LQFM182 (2) reduced the activity of the myeloperoxidase
enzyme, which is predominantly present in active neu-
trophils, confirming the previous result (Desmarchelier
et al. 1997; Eddouks et al. 2012).
Conflict of interest The authors declare that they have no conflicts of
interest.
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