Anti-inflammatory and antinociceptive activity profile of a new lead compound – LQFM219

Article abstract of DOI:10.1016/j.intimp.2020.106893

LQFM219 is a molecule designed from celecoxibe (COX-2 inhibitor) and darbufelone (inhibitor of COX-2 and 5-LOX) lead compounds through a molecular hybridisation strategy. Therefore, this work aimed to investigate the antinociceptive and anti-inflammatory activities of this new hybrid compound. The acute oral systemic toxicity of LQFM219 was evaluated via the neutral red uptake assay. Acetic acid-induced abdominal writhing and CFA-induced mechanical hyperalgesia were performed to evaluate the antinociceptive activity, and the anti-oedematogenic activity was studied by CFA-induced paw oedema and croton oil-induced ear oedema. Moreover, the acute anti-inflammatory activity was determined by carrageenan-induced pleurisy. In addition, cell migration, myeloperoxidase enzyme activity, and TNF-α and IL-1β levels were determined in pleural exudate. Moreover, a redox assay was conducted using electroanalytical and DPPH methods. The results demonstrated that LQFM219 was classified as GHS category 4, and it showed better free radical scavenger activity compared to BHT. Besides, LQFM219 decreased the number of writhings induced by acetic acid and the response to the mechanical stimulus in the CFA-induced mechanical hyperalgesia test. Furthermore, LQFM219 reduced oedema formation, cell migration, and IL-1β and TNF-α levels in the pleural cavity and inhibited myeloperoxidase enzyme activity. Thus, our study provides that the new pyrazole derivative, LQFM219, demonstrated low toxicity, antinociceptive and anti-inflammatory potential in vitro and in vivo.

Full text of DOI:10.1016/j.intimp.2020.106893

InternationalImmunopharmacology88(2020)106893
Contents lists available at ScienceDirect
International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
Anti-inflammatory and antinociceptive activity profile of a new lead
compound – LQFM219
Gustavo M. Galvãoa, Iziara F. Florentinob, Germán Sanzc, Boniek G. Vazc, Luciano M. Liãod,
José R. Sabinoe, Carina S. Cardosob, Daiany P.B. da Silvab, Elson A. Costab, Andreia L.P. Silvaf,
Artur C.G. da Silvaf, Marize C. Valadaresf, Jacqueline A. Leiteh, Eric de S. Gila,
⁎
Ricardo Menegattia,
a Laboratório de Química Farmacêutica Medicinal (LQFM), Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brazil
b Laboratório de Farmacologia de Produtos Naturais e Sintéticos, Departamento de Farmacologia, ICB, Universidade Federal de Goiás, Campus Samambaia, Goiânia, GO,
Brazil
c Laboratório de Cromatografia e Espectrometria de Massas - LaCEM, Instituto de Química, Universidade Federal de Goiás, Campus Samambaia, Goiânia, GO, Brazil
d Laboratório de Ressonância Magnética Nuclear, Instituto de Química, Universidade Federal de Goiás, Campus Samambaia, Goiânia, GO, Brazil
e Instituto de Física, Universidade Federal de Goiás, Campus Samambaia, Goiânia, GO, Brazil
f Laboratório de Farmacologia e Toxicologia Celular- FarmaTec, Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brazil
h Department of Pharmacology, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
LQFM219 is a molecule designed from celecoxibe (COX-2 inhibitor) and darbufelone (inhibitor of COX-2 and 5-
LOX) lead compounds through a molecular hybridisation strategy. Therefore, this work aimed to investigate the
antinociceptive and anti-inflammatory activities of this new hybrid compound. The acute oral systemic toxicity
of LQFM219 was evaluated via the neutral red uptake assay. Acetic acid-induced abdominal writhing and CFA-
induced mechanical hyperalgesia were performed to evaluate the antinociceptive activity, and the anti-oede-
matogenic activity was studied by CFA-induced paw oedema and croton oil-induced ear oedema. Moreover, the
acute anti-inflammatory activity was determined by carrageenan-induced pleurisy. In addition, cell migration,
myeloperoxidase enzyme activity, and TNF-α and IL-1β levels were determined in pleural exudate. Moreover, a
redox assay was conducted using electroanalytical and DPPH methods. The results demonstrated that LQFM219
was classified as GHS category 4, and it showed better free radical scavenger activity compared to BHT. Besides,
LQFM219 decreased the number of writhings induced by acetic acid and the response to the mechanical stimulus
in the CFA-induced mechanical hyperalgesia test. Furthermore, LQFM219 reduced oedema formation, cell mi-
gration, and IL-1β and TNF-α levels in the pleural cavity and inhibited myeloperoxidase enzyme activity. Thus,
our study provides that the new pyrazole derivative, LQFM219, demonstrated low toxicity, antinociceptive and
anti-inflammatory potential in vitro and in vivo.
Antinociceptive
Anti-inflammatory
2,3-di-tert-buthylphenol
IL-1β
TNF-α
Myeloperoxidase
1. Introduction
bradykinin, prostaglandins (PGs), leukotrienes, and cytokines [3], and
it occurs through the activation of a cascade of arachidonic acid via the
Inflammation is a normal and essential response, ranging from a
localized response to a generalized response, and is generally classified
as acute or chronic depending on the cellular and molecular events
involved [1,2]. The acute inflammatory response can be triggered by
tissue damage and/or infection that is recognised by resident macro-
phages via receptors of the innate immune system, such as family of the
Toll-like receptors (TLRs) [2]. This process is characterised by the
production and release of pro-inflammatory mediators, such as
action of cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome
P450 (CYP) enzymes [4].
The beginning of the inflammatory process occurs through the de-
livery of serotonin and histamine, which is followed by the recruitment
of polymorphonuclear (PMN) cells, among which neutrophils are the
most abundant. These cells are important for the elimination of pa-
thogens by releasing reactive oxygen species (ROS) and reactive ni-
trogen species present in their granules, as well as increasing the
⁎ Corresponding author.
E-mail address: rm_rj@yahoo.com (R. Menegatti).
https://doi.org/10.1016/j.intimp.2020.106893
Received 12 July 2020; Received in revised form 8 August 2020; Accepted 10 August 2020
1567-5769/©2020PublishedbyElsevierB.V.

G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
expression of myeloperoxidase (MPO), which is responsible for the
oxidation of pathogens [5,6]. The elimination of the pathogen is fol-
lowed by the phase of resolution and repair tissue by anti-inflammatory
mediators produced by resident macrophages. If the acute in-
flammatory response fails to eliminate the pathogen, the inflammatory
process persists and acquires a chronic characteristic, requiring phar-
macological intervention in many cases [7,8].
3 × 15 mL with CH2Cl2, and the combined organic layers were dried
with sodium sulphate and concentrated in vacuo. The crude product was
crystallised from (MeOH/H2O = 1:9) to 2,6-di-tert-butyl-4-((1-phenyl-
1H-pyrazol-4-yl)methyl)phenol (3) (181 mg, 50%) as a beige solid
(m.p. = 122–124 °C, Rf = 0.60 [hexene/AcOEt = 7:3]). IRmax (KBr)
cm−1: 3630 (ν O–H); 3093 (ν C–H); 2916 (ν C–H); 1599 (ν C = C); and
752 (ν Ar-1,4) (Fig. S1, Supplementary material); 1H NMR
(500.13 MHz) CDCl3 (δ): 7.57 (brs, H3); 7.68 (brd, J = 0.7 Hz, H5);
3.81 (s, H6); 7.65 (dd, J = 8.5; 1.2 Hz, H2′); 7.42 (ddd, J = 8.5; 7.5;
1.0 Hz, H3′); 7.24 (tt, J = 7.5; 1.2 Hz, H4′); 7.42 (ddd, J = 8.5; 7.5;
1.0 Hz, H5′); 7.65 (dd, J = 8.5; 1.2 Hz, H6′); 7.05 (s, H3″); 7.05 (s,
H5″); 1.43 (s, H8″); 5.08 (s, OH) (Fig. S2 and Table 2, Supplementary
material); 2D NMR (HSQC/HMBC-125.76 MHz) CDCl3/TMS (δ): 141.3
(C-3); 123.8 (C-4); 125.4 (C-5); 30.4 (C-6); 140.4 (C-1′); 118.7 (C-2′);
129.4 (C-3′); 126.0 (C-4′); 129.4 (C-5′); 118.7 (C-6′); 152.3 (C-1″);
136.1 (C-2″); 125.0 (C-3″); 131.1 (C-4″); 125.0 (C-5″); 136.1 (C-6″);
34.6 (C-7″); 30.4 (C-8″) (Figs. S3, S4; Table 2; Supplementary mate-
rial); [M + H]+m/z of 363.2385 (Fig. S5, Supplementary material).
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the
most used drugs in clinics worldwide due to their anti-inflammatory,
analgesic, and antipyretic properties. NSAIDs have a diversity of com-
pounds, with varied chemical structures that share similar therapeutic
and adverse effects. The main mechanism of action related to NSAIDs is
the inhibition of the cyclooxygenase enzymes (COX-1 and COX-2), thus
reducing the synthesis of PGs and thromboxane (TX). The prolonged
use of this class of drugs can cause damage to the gastrointestinal, renal,
and cardiovascular systems [9].
Based on this, this work aimed to design a new drug with anti-in-
flammatory and antioxidant properties, as well as improved safety and
tolerability. We describe the synthesis and biological evaluation of
LQFM219 (3), which was designed from celecoxibe (1) and darbufelone
lead compounds through a molecular hybridisation strategy. Celecoxibe
(1) is a COX-2 inhibitor, while darbufelone (2) is a dual inhibitor of
COX-2 and 5-LOX. To design LQFM219 (3), A and B scaffolds from
celecoxibe (1) and the C scaffold from darbufelone (2) were maintained
(Fig. 1). Our results demonstrated that LQF219 showed anti-in-
flammatory and antinociceptive activity through the reduction of pro-
inflammatory cytokines (IL-1 and IL-6) as well as the activity of the
MPO enzyme.
2.2. X-ray crystallography
Single crystals of LQFM219 (3) were obtained in acetone and se-
lected for x-ray diffraction data collection. A clear light-yellow, plate-
like specimen was mounted in a Bruker-AXS Kappa Apex II Duo dif-
fractometer operating with an IµS microsource of Cu-Kα radiation. The
raw data were processed using the APEX 2 suite of programs and cor-
rected for absorption effects using the multi-scan method with SADABS
[11]. The structure of the solution was obtained by direct methods
using SHELXS [12], and the final refinement was performed with full-
matrix least-squares on F2 using SHELXL [12]. The programs ORTEP-3
and SHELXS/SHELXL [12] were used in the WinGX [13] software
package.
2. Experimental
2.1. Chemicals
Synthesis of 2,6-di-tert-butyl-4-((1-phenyl-1H-pyrazol-4-yl)methyl)
phenol (3) (LQFM219) [10]: To a heated heterogeneous mixture of
formic acid (3 mL), 1-phenyl-1H-pyrazol-4-yl)methanol (7) (172 mg,
1 mmol), 2,6-di-tert-butylphenol (8) (206 mg, 1.00 mmol), and 2 mL of
CHCl3 were added, and the mixture was heated at 65 °C for 8 h. At the
end of the reaction, the residue was split between water and CH2Cl2.
The spelling formed was precipitate was filtered off under vacuum and
dried. The phases were separated, the aqueous layer was extracted
2.3. Electroanalytical assay
Voltammetric experiments were carried out with a potentiostat/
galvanostat (μAutolab III®) controlled with NOVA 2.1® software
(Metrohm). The measurements were obtained with a three-electrode
system consisting of a glassy carbon electrode, a Pt wire, and the Ag/
AgCl/KCl (3 M), representing the working electrode, the auxiliary
electrode, and the reference electrode, respectively. Assays were per-
formed in the potential range from 0 V to 1 V. The experimental con-
ditions for DPV analysis were as follows: pulse amplitude, 50 mV; pulse
width, 0.5 s; and scan rate, 10 mV s−1. The square wave voltammetry
(SWV) parameters were as follows: pulse of 50 mV, frequency of 50 Hz,
and potential increment of 2 mV, resulting in an effective scan rate of
100 mV s−1. The voltammograms were plotted with Origin8® software.
All experiments were conducted in triplicate (n = 3) in a 2.0 mL one-
compartment electrochemical cell at room temperature (21
1 °C),
and the main electrolyte used was 0.1 M phosphate buffer (PBS) (pH
7.0).
2.3.1. Electrochemical index
The electrochemical index (EI) considers parameters such as the
anodic peak potential (Epa), which is related to the electron donation
capacity, and the anodic peak current, which correlates with the ana-
lyte concentration (Ipa). Thus, since Epa is inversely proportional to the
ease of electron donation and Ipa is directly proportional to the elec-
troactive species concentration, EI is calculated using the following
equation:
n
Ipan
EI =
Epan
i=1
Fig. 1. Structural design of 2,6-di-tert-butyl-4-((1-phenyl-1H-pyrazol-4-yl)me-
thyl)phenol (LQFM219) (3) from celecoxibe (1) and darbufelone (2) lead
compounds.
where, in a voltammogram with n peaks, Ipan and Epan are the
current and potential, respectively, of the nth peak.
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G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
2.3.2. DPPH radical scavenging assay
2.5. Pharmacology
2.5.1. Animals
The radical scavenging assay was performed with DPPH reagent. A
mixture of 2.7 mL of DPPH solution with 0.3 mL methanol was pre-
pared, and the absorbance read at 517 nm was c.a. 0.7, which was used
as the control. In the assays, the 300 µL methanolic portion was sub-
stituted with the stock solution. The antioxidant capacity was expressed
as the IC50 value, representing the concentration (mM) of the sample
that produces 50% DPPH discoloration relative to the control solution.
All samples were read in a 1 cm glass recipient at room temperature
5 min after the stock solution transference to the recipient with DPPH.
All experiments were performed using adult male Swiss albino mice
(27–32 g) obtained from the Central Animal House of UFG. The animals
were maintained at constant room temperature (22
2 °C) under a
12 h light/dark cycle with free access to standard food and water. The
animals were acclimatised for 7 days before the start of the experi-
ments. Experimental groups of mice (n = 8) were used in this study. All
experimental protocols were developed according to the principles of
ethics and animal welfare designated by the Ethics Committee on
Animal Experimentation and approved by the Ethics Committee in
Research of UFG (number 17/13).
2.3.3. ABTS radical scavenging assay
The radical scavenging assay was performed with ABTS reagent. An
aqueous solution of 140 mM potassium persulfide was prepared and an
aqueous solution of 7 mM ABTS. Five millilitres of the ABTS solution
was mixed for each 84 µL of potassium persulfide solution, and the
mixture was left protected from light for 16 h. After this period, the
resulting solution was read, representing a mixture of 2.7 mL of ABTS
solution with 0.3 mL of methanol. Next, the absorbance read at 734 nm
was c.a. 0.7, which was used as a control. In the assays, the 300 µL
methanolic portion was substituted with the stock solution. The anti-
oxidant capacity was expressed as the IC50 value, representing the
concentration (mM) of the sample that produced 50% ABTS dis-
coloration relative to the control solution. All samples were read in a
1 cm glass recipient at room temperature 5 min after stock solution
transference to the recipient with ABTS.
2.5.2. Drugs and chemicals
The chemicals used in this study were acetic acid (Merck AG,
Darmstadt, Germany), DMSO (Sigma-Aldrich St. Louis, MO, USA),
carrageenan (Sigma-Aldrich, St. Louis, MO, USA), croton oil (Sigma
Chemical Co., St. Louis, MO, USA), dexamethasone (Decadron®, Ache,
Brazil), heparin (Cristália, SP, Brazil), hydrogen peroxide (Bioshop, GO,
Brazil), o-dianisidine (Sigma-Aldrich, St. Louis, MO, USA), sodium
azide (Sigma-Aldrich, St. Louis, MO, USA), Türk liquid (Bioshop, GO,
Brazil), and LQFM219 (3), which were dissolved in 10% DMSO in
distilled water. All other drugs were dissolved in distilled water. The
doses of the LQFM219 (3) were based on the doses of LQFM182 [16]
used in previous studies.
2.5.3. Anti-nociceptive activity
2.5.3.1. Acetic acid-induced abdominal writhing test. The acetic acid-
induced nociception was performed as previously described by Koster
et al. [17]. Groups of mice (n = 8) were treated by gavage (p.o.) with
vehicle (10% DMSO, 10 mL/kg) or LQFM219 (3) at doses of 25, 50, or
100 mg/kg or dexamethasone (1 mg/kg, positive control) 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
2.4. Toxicology
2.4.1. Cell culture
Balb/c 3 T3-A31 fibroblasts were obtained from the Rio de Janeiro
Cell Bank (Rio de Janeiro, RJ, Brazil). 3 T3 cells were cultured in
DMEM supplemented with 10% (v/v) heat-inactivated FBS, 100 IU/mL
penicillin, and 100 µg/mL streptomycin. The cell cultures were main-
tained at 37 °C in a humidified 5% CO2 atmosphere. The cells were
passaged three times a week and used in the exponential growth phase.
Cell viability was analysed using a TC20TM automated cell counter
(Hercules, CA, USA) according to the manufacturer’s instructions. A
value of > 90% was considered satisfactory for conducting the assays.
expressed as the mean
SD of the number of writhings.
2.5.3.2. CFA-induced mechanical hyperalgesia test. Experimental groups
of male mice (n = 8) were treated with vehicle (control, 10% DMSO,
10 mL/kg p.o.), LQFM219 (3) (100 mg/kg, p.o.), or dexamethasone
(1 mg/kg, positive control) 1 h before the injection of 20 µL of CFA
(1 mg/mL) in the right paw. The left paw, which received the same
volume of 0.9% (w/v) NaCl solution, was used as the control. The
nociceptive threshold in response to the mechanical stimulus was
measured from exposure of the inflamed and the non-inflamed hind
paw to an increasing force until the appearance of a nociceptive
reaction (vocalisation or paw withdrawal). The stimulation was
stopped when the mouse struggled to withdraw the corresponding
paw, and the threshold value was recorded. Measurements were
alternately performed for each paw. The cut-off for each animal was
450 g. The nociceptive threshold was evaluated by the differences
between the two paws (Δ) at 0, 2, 4, 8, and 24 h after CFA injection
using a Raldall Selito analgesimeter (Insight EFF-440, Brazil). The
baseline was established before the treatments (time 0) for each animal.
2.4.2. In vitro prediction of acute oral toxicity
Acute oral toxicity estimation in vitro was performed by evaluation
of the cytotoxicity of LQFM219 (3) using Balb/c 3T3 cells according to
ICCVAM [14]. Briefly, the Balb/c 3 T3-A31 cells (3 × 104 cells/well)
were seeded in 96-well plates overnight. The cells were then treated
with 8 different concentrations of LQFM219 (3) (200–12.5 µg/mL) for
48 h in an incubator (Thermo Scientific, USA) with a humidified at-
mosphere at 37 °C in 5% CO2. Next, the solutions were removed from
the plates, and 100 µL of DMEM containing 5% (v/v) FBS and neutral
red (NR, 0.25 mg/mL) was added to each well. After 3 h of incubation,
the NR medium was discarded, and the cells were washed with PBS.
The PBS was decanted from the plate, and 100 µL of NR desorb solution
(50 EtOH: 1 acetic acid: 49 water) was added to all wells. The plates
were shaken, and the absorbance was measured at 550 nm using a
spectrophotometer (Thermo Scientific Multiskans Spectrum, Boston,
MA, USA). Each concentration was tested in six replicates in three in-
dependent experiments. Cell viability was expressed as a percentage of
the control, and the IC50 value was obtained and used to estimate the
LD50 (median lethal dose) based on the following equation created with
the in-house model previously established by Vieira (2011) [15]: Log
(LD50) = 0.545 × log (IC50) + 0.757. The estimated LD50 value was
then used to classify the probable acute oral systemic toxicity of
LQFM219 (3), according to the Globally Harmonized Classification and
Labelling of Chemicals (GSH).
The results are expressed as the means
SD in grams [18,19].
2.5.4. Anti-inflammatory activity
2.5.4.1. CFA-induced paw oedema test. The paw oedema test was
performed as previously described by Passos et al. (2007) and
Nascimento et al. (2016) [20,21]. Experimental groups of male mice
(n = 8) were treated with vehicle (control, 10% DMSO, 10 mL/kg,
p.o.), LQFM219 (3) (100 mg/kg, p.o.), or dexamethasone (1 mg/kg,
positive control) 1 h before injection of 20 µL of CFA (1 mg/mL) in the
right paw. The left paw, which received the same volume of 0.9% (w/v)
NaCl solution, was used as the control. Oedema was then measured by
3

G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
Fig. 2. (A) Synthetic route used for the preparation of LQFM219 (3). (B) ORTEP view of the compound.
the difference in volume between the paws (paw with carrageenan and
saline) at different times (0, 2, 4, 8, and 24 h) after injection of CFA
using a plethysmometer (Ugo Basile Co., Italy).
transferred to a microplate well, and the absorbance was monitored
at
a
wavelength of 450 nm. The results are expressed as the
SD of enzymatic activity in mU/mL.
means
2.5.4.2. Croton oil-induced ear oedema test. Experimental groups of
male mice (n = 8) were treated with vehicle (control, 10% DMSO,
10 mL/kg, p.o.), LQFM219 (3) (100 mg/kg, p.o.), or dexamethasone
(1 mg/kg, positive control), and 60 min later cutaneous inflammation
was induced by applying 25 μL of a solution of croton oil in 2.5%
acetone (v/v) to the inner surface of the right ears of the mice. The same
volume of acetone was applied to the left ear. Four hours after croton
oil-induced inflammation, the mice were euthanised, and a 6-mm-
diameter plug was obtained from both treated and untreated ears with a
punch. The inflammatory response (oedema) was monitored by
measuring the difference in weight (mg) between the two plugs [22].
2.5.4.5. Measurement of cytokines. The pleural exudates of mice treated
with vehicle (10% [v/v] DMSO, 10 mL/kg, p.o.), LQFM219 (3)
(100 mg/kg, p.o.), or dexamethasone (1 mg/kg, positive control)
were also used to determine the concentrations of TNF-α and IL-1β
using an immunosorbent assay kit (ELISA) (Ebioscience). Samples were
collected 4 h after the induction of pleurisy and centrifuged at 1200 g
for 10 min at 4 °C, and the supernatant was separated and stored
(−70 °C) until the assay. The results are expressed as the means
in pg/mL [25,26].
SD
2.6. Statistical analysis
2.5.4.3. Carrageenan-induced pleurisy test. The pleurisy test was
performed as previously described by Saleh et al. (1999) [23].
Experimental groups of mice (n = 8) were treated with vehicle (10%
[v/v] DMSO, 10 mL/kg, p.o.), LQFM219 (3) (100 mg/kg, p.o.), or
dexamethasone (1 mg/kg, positive control) 1 h before injection of
100 µL of 1% carrageenan into the pleural cavity. The pleural exudate
was collected with 1 mL of heparinised PBS 4 h after carrageenan
administration. One aliquot was used to determine the total leukocyte
count using Türk liquid in a Neubauer’s chamber, and the other aliquot
was used to measure the MPO activity and cytokine levels.
All the results are expressed as the mean
SD The data were
analysed statistically by one-way ANOVA followed by Tukey’s post-hoc
test or by two-way ANOVA followed by Bonferroni’s post-hoc test.
Values of p ≤ 0.05 were considered significant. All statistical analyses
were carried out using Graph Pad Prism® version 5.00.
The assays were carried out in three independent experiments. The
cytotoxicity results, expressed as the mean
SD of six replicates, were
transformed into percentages of the control, and IC50 values were ob-
tained by non-linear regression.
3. Results
2.5.4.4. Myeloperoxidase (MPO) assay. MPO activity was measured as
previously described by Lino et al. (2013) [24]. Briefly, 40 µL of the
pleural exudate samples from the animals treated with vehicle (10% [v/
v] DMSO, 10 mL/kg, p.o.), LQFM219 (3) (100 mg/kg, p.o.), or
dexamethasone was added to 360 µL of phosphate buffer (pH 6.0)
containing 0.167 mg/mL of o-dianisidine, 2 HCl, and 0.0005% H2O2.
The enzymatic reaction was stopped after 15 min by the addition of
20 µL of 1% (w/v) sodium azide. The samples were subsequently
centrifuged for 5 min at 300 g. The supernatant (100 µL) was
3.1. Synthesis of 2,6-di-tert-butyl-4-((1-phenyl-1H-pyrazol-4-yl)methyl)
phenol - LQFM219 (3)
As illustrated in Fig. 2A, the synthetic route began with a con-
densation reaction between 1-(phenyl)hydrazine hydrochloride (4) and
1,1,3,3-tetramethoxy propane, as described by Finar and Hurlock [27],
to provide 1-(phenyl)-1-H-pyrazole (5) with a yield of 91%. The che-
moselective and regiospecific formylation of 1-(phenyl)-1-H-pyrazole
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G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
Table 1
Electrochemical index, DPPH, and ABTS IC50 of LQFM219 (3) and BHT.
EI (µA/V)
DPPH IC50 (mM)
ABTS IC50 (mM)
LQFM219 (3)
0.9211
0.7098
0.27
0.25
0.012*
0.012*
0.32
0.38
0.003*
0.004*
BHT
* Values given as the average
SD (n = 3).
(5) to 1-(phenyl)-1-H-pyrazole-4-carbaldehyde (6) was carried out
under Duff's conditions, with 84% yield [28]. In turn, the 1-(phenyl)-1-
H-pyrazole-4-carbaldehyde (6) was reduced to 1-phenyl-1H-pyrazol-4-
yl)methanol (7) through the use of NaBH4 in methanol, with a 91%
yield [29]. Finally, 1-phenyl-1H-pyrazol-4-yl)metanol (7) was reacted
with 2,6-di-tert-butylphenol (8) through a Friedel-Crafts alkylation re-
action using HCO2H as a catalyser to obtain 2,6-di-tert-butyl-4-((1-
phenyl-1H-pyrazol-4-yl)methyl)phenol (LQFM219) (3), with a yield of
50% [10]. LQFM219 (3) was obtained after four synthetic steps, with a
35% global yield.
Fig. 4. Cytotoxic activity of LQFM219 in murine fibroblast (BALB/3T3) cells
after 48
h of treatment with different concentrations of compound
(200–12.5 µg/mL). Cytotoxic effects were determined by the neutral red uptake
assay. The results represent the mean
in six replicates.
SD of three independent experiments
3.2. X-ray crystallography
The ORTEP view of LQFM219 (3) is illustrated in Fig. 2B, as well as
crystal data and structure refinement for LQFM219 (3) (Table 1,
Supplementary material).
processes are quasi-irreversible. Individual analysis of the EI and the
DPPH/ABTS IC50 calculated results showed that LQFM219 (3) had
better antioxidant capacity than 2,6-di-tert-butyl-4-methylphenol (BHT)
since higher EI values and lower IC50 infer higher antioxidant capacity
(Table 1).
3.3. Electroanalytical assay
In LQFM219 (3) voltammograms, it was possible to observe two
anodic peaks: Epa1 c.a. 0.05 V and Epa2 c.a. 0.5 V (Fig. 3A). In the
cyclic voltammetry (Fig. 3B), only the major second anodic peak, Epa2
c.a. 0.5 V, was observed, which demonstrated no signs of reversibility.
Slight reversibility was observed for both oxidative processes (Fig. 3C);
however, due to their amplitude, it is safe to assume that these
3.4. In vitro assay to estimate the LD50 value
The cytotoxic effects of the eight concentrations of LQFM219 (3)
(200–12.5 µg/mL) on Balb/c 3T3-A31 cells determined by the NRU
assay are shown in Fig. 4. LQFM219 (3) inhibited cell proliferation in a
concentration-dependent manner. Using these findings as a basis, the
Fig. 3. Differential pulse (A), cyclic (B), and square wave (C) voltammograms of a 1 mM LQFM219 stock solution in PBS (pH 7.0) with glassy carbon.
5

G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
Fig. 5. Effect of LQFM219 (3) (25, 50, and 100 mg/kg, p.o.) on the number of
acetic acid-induced writhings in mice (n = 8). Vertical bars represent the
mean
SD of the number of writhings in 30 min for each experimental group.
Dexamethasone (Dexa, 1 mg/kg) was used as a positive control. *p ≤ 0.05,
**p ≤ 0.01, and ***p ≤ 0.001 (compared with the control group), according to
ANOVA followed by Tukey’s test.
Fig. 6. Antihyperalgesic effect of LQFM219 (3) (100 mg/kg, p.o.) and dex-
amethasone (Dexa, 1 mg/kg; p.o., positive control) on the CFA-induced me-
chanical hyperalgesia test in mice (n = 8). Treatment with LQFM219 (3) and
dexamethasone began to reduce mechanical hyperalgesia starting from hour 2
until hour 24 after CFA injection. The values are expressed as the mean
SD
LD50 was estimated to be 369.1 mg/kg. Therefore, LQFM219 (3) was
of the difference between non-inflamed and inflamed paws, in grams (g).
*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, compared with the control group,
according to two-way ANOVA followed by Bonferroni's post-hoc test.
classified as GHS category 4 (300 mg/kg > LD50 < 2000 mg/kg).
3.5. Anti-nociceptive activity
paw oedema formation. At hour 2 of the test, compared with the control
3.5.1. Acetic acid-induced abdominal writhing test
group (62.5
7.1 μL), treatments with LQFM219 (3) and with dex-
To evaluate the possible antinociceptive activity of LQFM219 (3),
the acetic acid-induced abdominal writhing test was performed. This
test is a visceral pain model that is widely employed and has been
frequently used to evaluate peripheral antinociceptive activity [30].
The oral treatments with LQFM219 (3) at doses of 25, 50, or 100 mg/kg
decreased the number of writhings induced by acetic acid in a dose-
dependent manner, compared with the control group (vehicle, 10%
amethasone (1 mg/kg) reduced oedema by 46.0% (P ≤ 0.01) and
62.0% (P ≤ 0.001), respectively. At hour 4, compared with the control
group (93.7
15.1 μL), oedema was reduced by 45.3% and 65.3%
(P ≤ 0.001), respectively. At hour 8, compared with the control group
(125 20.7 μL), oedema was reduced by 27.0% (P ≤ 0.05) and 68%
(P ≤ 0.001), respectively. At hour 24, compared with the control group
(177.5
15.8 μL), oedema was reduced by 33.1% (P ≤ 0.05) and
DMSO, 10 mL/kg), from 97.4
8.7 (group treated with vehicle) to
71.1% (P ≤ 0.001), respectively (Fig. 7).
84.0
62.7
5.0 (13.7%, P ≤ 0.05), 79.6
10.1 (18.2%, P ≤ 0.01), and
8.9 (35.6%, P ≤ 0.001), respectively. The positive control for
this test, dexamethasone (1 mg/kg), reduced the number of writhings
3.6.2. Croton oil-induced ear oedema test
by 58.4
7.9 (40.1%, P ≤ 0.001) (Fig. 5).
Croton oil contains 12-O-tetradecanoylphorbol-13-acetate (TPA)
and other phorbol esters as main irritant agents. In this test, oral
treatments with LQFM219 (3) (100 mg/kg) reduced ear oedema for-
mation by 57.6% (P ≤ 0.001) compared with the control group
3.5.2. CFA-induced mechanical hyperalgesia test
The treatment with LQFM219 (3) (100 mg/kg) reduced the differ-
ence in the nociceptive threshold between the non-inflamed and in-
flamed paw of animals in response to a mechanical stimulus con-
sistently in the paw pressure test. At hour 2 of the test, compared with
(13.4
3.3 mg). The positive control, dexamethasone (1 mg/kg),
reduced ear oedema by 78.3% (P ≤ 0.001), as shown in (Fig. 8).
the control group (137.5
32.7 g), treatments with LQFM219 (3)
3.6.3. Carrageenan-induced pleurisy test
(100 mg/kg) and with dexamethasone (1 mg/kg) reduced mechanical
hyperalgesia by 35.8% and 55.7% (P ≤ 0.001), respectively. At hour 4
Injection of carrageenan into the pleural cavity of mice induced an
acute inflammatory response characterised by the accumulation of
leukocytes. Oral treatments with LQFM219 (3) (100 mg/kg) and with
dexamethasone (1 mg/kg) reduced the total number of leukocytes to
of the test, compared with the control group (201.9
22.9 g), treat-
ments with LQFM219 (3) (100 mg/kg) and with dexamethasone (1 mg/
kg) reduced mechanical hyperalgesia by 70.3% and 81.4% (P ≤ 0.001),
respectively. At hour 8 of the test, compared with the control group
3.42
1.7
0.9 leukocytes × 106/mL (31.3% reduction, P ≤ 0.001) and
0.5 leukocytes × 106/mL (65.1% reduction, P ≤ 0.001), re-
(183.6
31.1 g), treatments with LQFM219 (3) (100 mg/kg) and with
spectively, when compared to the control group (5.0
0.6 leuko-
dexamethasone (1 mg/kg) reduced mechanical hyperalgesia by 21.2%
(P ≤ 0.05) and 31.2% (P ≤ 0.001), respectively. At hour 24 of the test,
cytes × 106/mL), as shown in (Fig. 9A).
compared with the control group (174.6
33.0 g), treatments with
LQFM219 (3) (100 mg/kg) and with dexamethasone (1 mg/kg) reduced
mechanical hyperalgesia by 23.2% (P ≤ 0.05) and 47.2% (P ≤ 0.001),
respectively (Fig. 6).
3.6.4. Myeloperoxidase (MPO) assay
In the carrageenan-induced pleurisy test, LQFM219 (3) (100 mg/kg)
also decreased myeloperoxidase activity to 75.4
7.7 mU/mL (69.0%
0.001) compared with the control group
65.6 mU/mL). A similar result was observed for the positive
reduction,
(242.9
P
≤
3.6. Anti-inflammatory activity
control, dexamethasone (1 mg/kg, p.o.), which resulted in a decrease to
3.6.1. CFA-induced paw oedema test
117.7
28.4 mU/mL (51.5% reduction, P ≤ 0.001), as shown in
Treatments with LQFM219 (3) (100 mg/kg) consistently reduced
(Fig. 9B).
6

G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
profile of a new compound, LQFM219 (3), which was designed from
celecoxibe (1) and darbufelone (2) using a molecular hybridisation
strategy. The new compound, LQF219, showed an anti-inflammatory
and antinociceptive effect that involves a reduction in the oedema in-
duced by different phlogistic agents, a decrease in the levels of pro-
inflammatory cytokines, and inhibition of the migration of poly-
morphonuclear cells in carrageenan-induced pleurisy.
Initially, the toxicity of the new compound, LQFM219 (3), was de-
termined in vitro. LQFM219 (3) was classified as GHS category 4
(300 < LD50 < 2000 mg/kg), whereas darbufelone (1) showed a
greater potential to promote acute oral toxicity since it was classified as
GHS category 3 [4]. According to OECD, chemicals classified under
GHS category 4 have low/moderate acute toxicity potential [31]. The
results obtained for the cytotoxicity assay allowed us to estimate the
DL50 for compound LQFM219 (3) as 369.1 mg/kg, demonstrating that
this compound may be useful for the development of new drugs.
The acetic acid model was used to assess the antinociceptive activity
of LQFM219 since it induces pain indirectly via the release of some
endogenous mediators, such as prostaglandins and cytokines that re-
duce the threshold of nociception and stimulate nociceptive fibres [32].
In this test, oral treatments with LQFM219 (3) at doses of 25, 50 or
100 mg/kg decreased the number of writhings induced by acetic acid in
a dose-dependent manner. Moreover, LQFM219 (3) at a dose of
100 mg/kg significantly inhibited the mechanical hyperalgesia asso-
ciated with CFA. CFA induced inflammatory pain by regulating NF-κB
activation and increases in PGE2, TNF-α, IL-1β, and IL-6, which are
capable of sensitising nociceptive neurons, favouring hyperalgesia
[33,34]. We used dexamethasone as a positive control in the experi-
ment since several studies have reported its potential to reduce in-
flammatory pain [35–37]. As expected, dexamethasone treatment re-
duced the CFA-induced pain at all times measured. Therefore, these
data suggest that part of the antinociceptive effect might be associated
with a possible anti-inflammatory activity of LQFM219 (3). Thus, we
investigated the anti-inflammatory activity of LQFM219 (3) by CFA-
induced paw oedema and croton oil-induced ear oedema.
Fig. 7. Anti-oedematogenic effect of LQFM219 (3) (100 mg/kg, p.o.) and
dexamethasone (Dexa, 1 mg/kg; p.o., positive control) on the CFA-induced
oedema test in mice (n = 8). Treatment with LQFM219 (3) and dexamethasone
began to reduce oedema starting from hour 2 until hour 24 after CFA injection.
The values are expressed as the mean
SD of the difference between paws, in
μL. ***p ≤ 0.001, compared with the control group, according to two-way
ANOVA followed by Bonferroni's post-hoc test.
CFA and croton oil produce acute inflammation by a local release of
mediators, such as cytokines and prostaglandins, which are involved in
the formation of oedema, leukocyte migration, vasodilation, and hy-
peralgesia [38–41]. LQFM219 (3) was able to inhibit CFC oedema
formation 24 h after the stimulus. A similar effect was observed for
CFA-induced hyperalgesia. In addition, at a dose of 100 mg/kg,
LQFM219 (3) inhibited ear oedema formation induced by croton oil. As
expected, treatment with dexamethasone (1 mg/kg), the positive con-
trol, inhibited the oedema induced by CFA and croton oil. Together,
these data demonstrate the antiedematogenic effect of the new com-
pound LQFM219 (3). The results corroborate some previous studies,
which indicates antinociceptive and anti-inflammatory activities of
pyrazole derivatives [24,42–44]. Furthermore, Lino and collaborators
(2017) demonstrated anti-inflammatory and antinociceptive activities
for the compound LQFM091, derived from darbufelone [45].
Fig. 8. Anti-oedematogenic effect of LQFM219 (3) (100 mg/kg, p.o.) and
dexamethasone (Dexa, 1 mg/kg; p.o., positive control) on croton oil-induced
ear oedema in mice. Values are the mean
SD (n = 8) of differences in
weight between right and left ear plugs. ***p ≤ 0.001, compared with the
control group, according to ANOVA followed by Tukey’s test.
3.6.5. Measurement of cytokines
To investigate the action of LQFM219 (3) on different parameters of
the inflammatory process, the carrageenan-induced pleurisy test was
performed. Carrageenan-induced pleurisy is a model extensively used
to assess leukocyte recruitment, especially neutrophils. Different studies
have shown an accumulation of neutrophils 4 h after injection with
carrageenan in the pleural region, as well as the production and release
of mediators that play important to inflammatory response [25,46–48].
Our data demonstrated that treatment with LQFM219 (3) (100 mg/kg)
reduced leukocyte migration. It is known that MPO is more abundant in
immune cells, such as neutrophils, and the level of activity is directly
proportional to the neutrophil concentration in inflamed biological li-
quids or tissues [49,50]. Consequently, we performed the MPO activity
and observed that LQFM219 (3) reduced MPO activity, suggesting that
the action of the new compound could be due to a preferential reduc-
tion in neutrophils or a direct inhibition of MPO enzyme activity.
The pro-inflammatory cytokines are involved in cellular
In addition to the effect of LQFM219 (3) on leukocyte counts, we
observed that this compound also caused significant changes in the
production/release of some pro-inflammatory cytokines. In our ex-
periments, LQFM219 (3) (100 mg/kg) caused a significant reduction in
TNF-α levels (percent inhibition of 54.0%, P ≤ 0.001) compared with
the control group (18.2
5.7 pg/mL) (Fig. 10A). The same dose also
reduced IL-1β levels (percent inhibition of 48.4%, P ≤ 0.01) compared
with the control group (122.1
50.4 pg/mL) (Fig. 10B). As expected,
dexamethasone (1 mg/kg) showed the expected profile through in-
hibition of pro-inflammatory cytokines (percent inhibition on TNF-α of
88.2% and IL-1β of 95.5%, P ≤ 0.001) (Fig. 10A, B).
4. Discussion
The present study reported the synthesis and pharmacological
7

G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
Fig. 9. Anti-inflammatory effect of LQFM219 (3) (100 mg/kg, p.o.) and dexamethasone (Dexa, 1 mg/kg; p.o., positive control) on the carrageenan-induced pleurisy
test in mice. (A) Leukocyte counts in the pleural cavity. (B) Myeloperoxidase enzyme activity (mU/mL). Values are expressed as the mean
SD of 8 mice.
***p ≤ 0.001, compared with the carrageenan group, ###p ≤ 0.001 (Saline × Carrageenan), according to ANOVA followed by Tukey’s test.
recruitment, acute phase protein release, increases in vascular perme-
ability, and hyperalgesia, in addition to inducing the production of
other pro-inflammatory mediators, contributing to the progression of
inflammation [51–54]. Thus, we analysed the levels of IL-1β and TNF-α
in the pleural exudate after the challenge with carrageenan. The
treatment with LQFM219 (3) markedly reduced the concentrations of
IL-1β and TNF-α in the pleural exudate, suggesting that the reduction of
cell migration and antinociceptive effect observed was due, in part, to
the reduction of these pro-inflammatory cytokines.
has antinociceptive, anti-inflammatory, and antioxidant activities. The
anti-inflammatory activity was characterised by a reduction of leuko-
cyte migration in the pleural exudate, associated with inhibition of
MPO activity, reduced levels of TNF-α and IL-1β, and free radical
scavenger activity, demonstrating that this compound may be useful for
the development of new analgesic and anti-inflammatory drugs.
CRediT authorship contribution statement
Oxidative stress plays a critical role in acute inflammatory re-
sponses, such as lung injury [55] and several preclinical studies have
shown that compounds with antioxidant effects also have considerable
anti-inflammatory activity [56,57]. Therefore, we investigated the
possible antioxidant effect of LQFM219 (3). As we demonstrated in
cyclic voltammetry assays, LQFM219 (3) showed better antioxidant
capacity than BHT, a gold standard antioxidant that is commonly used
in the foods, pharmaceuticals, petroleum products, rubber, and oil in-
dustries. These findings indicate that the presence of BHT in the che-
mical structure of LQFM219 (3) may have contributed positively to the
anti-inflammatory effect demonstrated by this compound.
Gustavo M. Galvão: Methodology, Investigation, Data curation,
Writing
- original draft. Iziara F. Florentino: Methodology,
Investigation, Data curation. Germán Sanz: Methodology,
Investigation. Boniek G. Vaz: Formal analysis, Supervision. Luciano
M. Lião: Formal analysis, Supervision. José R. Sabino: Formal ana-
lysis, Supervision. Carina S. Cardoso: Methodology, Investigation.
Daiany P.B. da Silva: Methodology, Investigation, Data curation.
Elson A. Costa: Formal analysis. Andreia L.P. Silva: Methodology,
Investigation. Artur C.G. da Silva: Methodology, Investigation, Data
curation, Writing - original draft. Marize C. Valadares: Formal ana-
lysis, Supervision. Jacqueline A. Leite: Supervision. Eric de S. Gil:
Formal analysis, Supervision. Ricardo Menegatti: Conceptualization,
Project administration, Supervision.
5. Conclusion
In conclusion, the present findings show, for the first time, that
LQFM219 (3), a molecule designed from celecoxibe and darbufelone,
Fig. 10. Effect of LQFM219 (3) (100 mg/kg, p.o.) and dexamethasone (Dexa, 1 mg/kg; p.o., positive control) on cytokine levels. (A) Tumor necrosis factor-alpha
(TNF-α) levels. (B) Interleukin 1β (IL-1β) levels. Vertical bars represent the mean
SD of 8 mice. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, compared with the
control group, ###p ≤ 0.001 (saline × carrageenan), according to ANOVA followed by Tukey’s test.
8

G.M. Galvão, et al.
InternationalImmunopharmacology88(2020)106893
Acknowledgments
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