Design, synthesis and pharmacological assessment of new pyrazole compounds

Article abstract of DOI:10.1007/s10787-020-00727-1

Aims: This study investigated the antinociceptive and anti-inflammatory effects of new pyrazole compounds LQFM011(5), LQFM043(6) and LQFM044(7) as well as the mechanisms of action and acute in vitro toxicity. Main methods: The antinociceptive activity was evaluated using the acetic acid-induced abdominal writhing test, formalin-induced pain test and the Randall–Selitto test. The anti-inflammatory activity was evaluated using models of paw oedema and pleurisy induced by carrageenan; cell migration, the levels of tumour necrosis factor α (TNF-α) and myeloperoxidase (MPO) enzyme activity were evaluated. In addition, the ability to inhibit phospholipase A2 (PLA2) in vitro and docking in PLA2 were used. Acute oral systemic toxicity in mice was evaluated through the neutral red uptake assay. Key findings: The synthesised compounds (5–7), delivered via gavage (p.o.) at 70, 140 or 280 μmol/kg, decreased the number of writhings induced by acetic acid; the three compounds (280 μmol/kg p.o.) reduced the paw licking time in the first and second phase of the formalin test and decreased the nociceptive threshold variation in the Randall–Selitto test. Furthermore, this dose reduced oedema formation, leucocyte migration (specifically through reduction in polymorphonuclear cell movement) and increased mononuclear cells. MPO activity and the levels of pro-inflammatory cytokines TNF-α were decreased. Evaluation of PLA2 inhibition via the docking simulation revealed more interactions of LQFM043R(6) and LQFM044(7), data that corroborated the half-maximal inhibitory concentration (IC₅₀) of PLA2 inhibition in vitro. Therefore, LQFM011(5), LQFM043(6) and LQFM044(7) were classified with the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) as category 4.

Full text of DOI:10.1007/s10787-020-00727-1

Infammopharmacology
Inflammopharmacology
https://doi.org/10.1007/s10787-020-00727-1
ORIGINAL ARTICLE
Design, synthesis and pharmacological assessment of new pyrazole
compounds
Jordana C. Oliveria1 · Daiany P. B. Silva6 · Iziara F. Florentino6 · Lidya C. da Silva2 · Germán Sanz2 · Boniek G. Vaz2 ·
Francine Pazini3 · Flávio S. de Carvalho1 · Luciano M. Lião4 · Thaís Rosa Marques dos Santos5 · Marize C. Valadares5 ·
Elson A. Costa6 · Fernanda Cristina Alcantara dos Santos7 · Bianca Villavicencio8 · Hugo Verli8 · Ricardo Menegatti1
Received: 5 March 2020 / Accepted: 29 May 2020
© Springer Nature Switzerland AG 2020
Abstract
Aims This study investigated the antinociceptive and anti-infammatory efects of new pyrazole compounds LQFM011(5),
LQFM043(6) and LQFM044(7) as well as the mechanisms of action and acute in vitro toxicity.
Main methods The antinociceptive activity was evaluated using the acetic acid-induced abdominal writhing test, formalin-
induced pain test and the Randall–Selitto test. The anti-infammatory activity was evaluated using models of paw oedema
and pleurisy induced by carrageenan; cell migration, the levels of tumour necrosis factor α (TNF-α) and myeloperoxidase
(MPO) enzyme activity were evaluated. In addition, the ability to inhibit phospholipase A2 (PLA2) in vitro and docking in
PLA2 were used. Acute oral systemic toxicity in mice was evaluated through the neutral red uptake assay.
Key fndings The synthesised compounds (5–7), delivered via gavage (p.o.) at 70, 140 or 280 µmol/kg, decreased the number
of writhings induced by acetic acid; the three compounds (280 µmol/kg p.o.) reduced the paw licking time in the frst and
second phase of the formalin test and decreased the nociceptive threshold variation in the Randall–Selitto test. Furthermore,
this dose reduced oedema formation, leucocyte migration (specifcally through reduction in polymorphonuclear cell move-
ment) and increased mononuclear cells. MPO activity and the levels of pro-infammatory cytokines TNF-α were decreased.
Evaluation of PLA2 inhibition via the docking simulation revealed more interactions of LQFM043R(6) and LQFM044(7),
data that corroborated the half-maximal inhibitory concentration (IC50) of PLA2 inhibition in vitro. Therefore, LQFM011(5),
LQFM043(6) and LQFM044(7) were classifed with the Globally Harmonized System of Classifcation and Labelling of
Chemicals (GHS) as category 4.
Keywords Anti-infammatory · Pyrazole · TNF-α · PLA2
5
* Ricardo Menegatti
Laboratory of Pharmacology and Cell Toxicology, Faculty
of Pharmacy, Federal University of Goiás, Goiânia, GO,
Brazil
rm_rj@yahoo.com
1
Laboratory of Medicinal Pharmaceutical Chemistry, College
6
7
8
Laboratory of Pharmacology of Natural and Synthetic
Products, Department of Pharmacology, Institute
of Biological Sciences, Federal University of Goiás, Goiânia,
GO, Brazil
of Pharmacy, Federal University of Goiás, Goiâniam, GO,
Brazil
2
Laboratory of Chromatography and Mass Spectrometry
(LaCEM), Chemistry Institute, Federal University of Goiás,
Laboratory of Microscopy Applied to Reproduction,
Department of Histology, Embryology and Cell Biology,
Institute of Biological Sciences, Federal University of Goiás,
Goiânia, GO, Brazil
Goiânia, GO, Brazil
3
Institute of Health Sciences, Federal University of Mato
Grosso, Sinop, MT, Brazil
4
Laboratory of Nuclear Magnetic Resonance, Chemistry
Center of Biotechnology, Federal University of Rio Grande
Institute, Federal University of Goiás, Goiânia, GO, Brazil
Do Sul, Porto Alegre, RS, Brazil
Vol.:(0123456789)
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J. C. Oliveria et al.
In the scope of a research programme aimed at drug
development for infammatory diseases, we describe in the
present study the design, synthesis and pharmacological
evaluation of new pyrazoles LQFM011(5), LQFM043(6) and
LQFM044(7). These compounds were originally designed
through a bioisosterism strategy from LQFM020(3) and
LQFM039(4) lead compounds. Specifcally, the fuorine
scafold present in LQFM020(3) (Oliveira et al. 2017) and
LQFM039(4) (Florentino et al. 2019) of the trifuoromethyl
scafold in LQFM043(6) and LQFM044(7) were changed.
On the other hand, the hydrogen scafold in LQFM011(5),
to investigate the electronic efect and steric hindrance of
the trifuoromethyl scafold present in LQFM043(6) and
LQFM044(7), were maintained.
Introduction
The infammatory process is benefcial and essential for
tissue homeostasis for various harmful conditions. It is
characterised by fve classical signs of infammation: pain,
heat, redness, swelling and loss of function (Lawrence and
Gilroy 2007; Medzhitov 2010). The process can be clas-
sifed according to the duration. Acute indicates a short
duration and with participation of the innate immune
system followed by healing and infammation; chronic is
of a longer duration and mediated by persistent infam-
mation. During this process, several pro-infammatory
mediators are released—cytokines [e.g. tumour necrosis
factor α (TNF-α)], prostaglandins (PGE2) and chemokines
(Medzhitov 2010)—into infamed tissue. This release pro-
motes vasodilation, cell migration due to rapid infux of
cells, principally polymorphonuclear cells (Barnig and
Levy 2015; Junior and Junior 2016), enzymatic activation
and sensitisation of nociceptive fbres (Junior and Junior
2016). The latter is responsible for infammatory pain, and
an important chemical mediator responsible for sensitisa-
tion and potentiation of nociceptive fbres is the PGE2
derivative of the arachidonic acid cascade. The arachi-
donic acid cascade begins with the catalysis of fatty acyl
ester bond at the sn-2 position of glycerophospholipids by
phospholipase A2 (PLA2) that release arachidonic acid
and is biotransformed by diferent enzymes, among them
cyclooxygenase (COX) and lipoxygenase (Dennis 1983).
PLA2 has an important role in the infammatory process,
pain, airway diseases and atherosclerosis; thus, this target
is of interest for the development of anti-infammatory
drugs (Murakami et al. 2011).
Materials and methods
Drugs and chemicals
Acetic acid (Merck, USA), dimethyl sulfoxide (DMSO;
Sigma-Aldrich, St. Louis, MO, USA), carrageenan (Sigma-
Aldrich), dexamethasone (Decadron®, Ache, Brazil), indo-
methacin (Indocid®, Merck Sharp & Dohme Farmacêu-
tica-Ltd, SP, Brazil), morphine hydrochloride (Dimorf®,
Cristalia, SP, Brazil) and ethylenediaminetetraacetic acid
(EDTA) (Sigma-Aldrich) were obtained from the indicated
vendors. Acetone, formaldehyde, heparin, NaCl and Türk
solution were procured from Isofar (Brazil), Synth (Brazil),
Hipolabor (Brazil), Belga (Brazil) and Bioshop (Brazil),
respectively. LQFM011(5), LQFM043(6) and LQFM044(7)
were dissolved in 10% DMSO in distilled water, and all other
drugs were dissolved only in distilled water. Dulbecco’s
Modifed Eagle Medium (DMEM), heat-inactivated foetal
bovine serum (FBS), streptomycin/penicillin solution and
trypsin, were purchased from Sigma-Aldrich.
Nonsteroidal anti-infammatory drugs (NSAIDS) and
cyclooxygenase (COX-1 and COX-2) inhibitors are the
most commonly used drugs to modulate infammation, but
long-term ingestion can cause side efects such as gastroin-
testinal ulcers and kidney and liver damage, and selective
COX-2 inhibitors are associated with cardiovascular side
efects (Wong 2019). Pyrazolic compounds present several
pharmacological activities, including anti-infammatory
and antinociceptive, with reductions in pro-infammatory
mediators (Florentino et al. 2017), TNF-α, cellular migra-
tion (Cidade et al. 2016) and modulation of the activation
of acid-sensitive ion channel 1 (ASIC-1), transient receptor
potential (TRPV1), and μ-xopiod receptor (MOR), to treat
pain (Florentino et al. 2019). TNF-α is a crucial infamma-
tory mediator because it induces the expression of COX, the
enzyme responsible for the synthesis of prostaglandins such
as PGE2 by the activation of the transcription factor nuclear
factor kappa B (NF-κβ) and can regulate the production of
other pro-infammatory cytokines such as interleukin (IL)-6
and IL-1β (Lawrence 2009; Liu et al. 2017).
Animals
All experiments were performed using female Swiss albino
mice (25–30 g) from the Central Animal House of the Fed-
eral University of the State of Goiás (UFG). The animals
were kept in plastic cages (41×34×16 cm) at constant room
temperature (22 2 °C) with access to food pellets and water
ad libitum and on a 12-h light/12-h dark cycle, in compli-
ance with the International Guiding Principles for Bio-
medical Research Involving Animals. All animal protocols
complied with the national and international laws regarding
animal care and were approved by the Local Ethics Commit-
tee on Animal Research by the Ethics Commission of UFG
(number 020/18).
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Design, synthesis and pharmacological assessment of new pyrazole compounds
Antinociceptive activity
Anti‑infammatory activity
Acetic acid‑induced abdominal writhing test
Carrageenan‑induced paw oedema
The acetic acid-induced abdominal writhing test was per-
formed as described previously by Koster et al. (1959).
The groups of animals (n = 8) were treated by gavage
(p.o.) with vehicle (10% DMSO 10 mL/kg), LQFM011(5),
LQFM043(6) or LQFM044(7) at doses of 70, 140 or
280 µmol/kg or with indomethacin (28 µmol/kg, posi-
tive control). After 1 h, the acetic acid solution [1.2%
v/v; 10 mL/kg, intraperitoneal (i.p.)] was applied into the
peritoneal cavity of the animals. Each was observed indi-
vidually, and the number of writhings were counted for
30 min. The results are expressed as number of writhings
and percentage reduction in the number of writhings.
The paw oedema test was used as the animal model of acute
infammation according to the method described by Winter
et al. (1962). The experimental groups of animals (n = 8)
were treated orally with vehicle (control, 10% DMSO,
10 mL/kg), LQFM011(5), LQFM043(6) or LQFM044(7)
(280 µmol/kg p.o.) or dexamethasone (3.8 µmol/kg p.o.,
positive control), 1 h before injection of 50 µL of 1% car-
rageenan into the right paw. The left paw, which received
the same volume of a 0.9% NaCl solution, was used as the
control, and the paw volume was measured using a plethys-
mometer (Ugo Basile Co., Italy) at diferent times (basal and
1, 2, 3 and 4 h). The oedema was evaluated by the diference
in the volume between the paws (paw with carrageenan and
saline).
Formalin test
Carrageenan‑induced pleurisy
The formalin-induced nociception test was conducted
according to the method described by Hunskaar and Hole
(1987). The groups of animals (n = 8) were treated with
vehicle (10% DMSO, 10 mL/kg p.o.), LQFM011(5),
LQFM043(6) or LQFM044(7) (280 µmol/kg p.o.), indo-
methacin (28 µmol/kg p.o., positive control in the sec-
ond phase) or morphine [7.4 µmol/kg subcutaneous (s.c.),
positive control in both phases]. One hour after the oral
treatment or 30 min after the subcutaneous treatment, the
mice received 20 µL of 3% formalin in the plantar surface
of the right hind paw. The licking time was assessed in two
phases: 0 to 5 min (frst phase) and 15 to 30 min (second
phase).
The pleurisy test was performed as described previously
by Lino et al. (2013). The experimental groups of animals
(n=8) were treated orally with vehicle (control, 10% DMSO,
10 mL/kg), 280 µmol/kg LQFM011(5), LQFM043(6) or
LQFM044(7) or 3.8 µmol/kg dexamethasone (positive con-
trol). One hour after the treatments, the animals received
an injection of 100 µL of 1% carrageenan into the pleu-
ral cavity. The pleural exudate was collected with 1 mL of
heparinised phosphate-bufered saline (PBS) 4 h after the
carrageenan injection. One aliquot was used to determine
the total leucocyte content using Türk liquid in a Neubauer
chamber, and other aliquots were used for the diferential
counts of mononuclear and polymorphonuclear leucocytes
(by optical microscopy), determination of myeloperoxidase
(MPO) activity and measurement of TNF-α by enzyme-
linked immunosorbent assay (ELISA). The results of the
total counts are expressed as the absolute number of total
cells, and diferential counts are presented for each cell type.
The result was also adjusted according to the volume unit
used in the performed evaluation and expressed as the num-
ber of leucocytes (106)/mL.
Randall–Selitto paw pressure test (mechanical
hyperalgesia)
Experimental groups of mice (n = 8) were treated
with vehicle (control, 10% DMSO, 10 mL/kg p.o.),
LQFM011(5), LQFM043(6), LQFM044(7) (280 µmol/kg
p.o.) or dexamethasone (3.8 µmol/kg, positive control) 1 h
before the injection of 50 µL of 1% carrageenan into the
right paw. The left paw received the same volume of 0.9%
(w/v) NaCl solution and was used as the control. Measure-
ments were alternately performed on each paw; the cut-of
for each animal was 450 g. The nociceptive threshold was
evaluated by diferences between the two paws (Δ) at 0, 1,
2, 3 and 4 h after the carrageenan injection, using a Ran-
dall–Selitto analgesimeter (Insight EFF-440, Brazil). The
baseline was performed before the treatments (time 0) for
each animal (Randall and Selitto 1957).
Histology
Immunohistochemistry and quantifcation of TNF‑α
immunoreactivity
Five animals from the paw oedema test were selected to
be sacrifced, and fragments of the hind paw were taken.
Briefy, the fragments were fxed in 4% paraformaldehyde
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J. C. Oliveria et al.
in 0.1 M phosphate bufer (pH 7.2) for 24 h or in methacarn
(60% methanol, 30% chloroform and 10% acetic acid) for
4 h. After this time, the fragments were washed in water,
dehydrated in a series of increasing ethanol concentrations,
clarifed in xylol and then embedded in parafn (26 Para-
plast, Histosec, Merk). The tissues were sectioned at 5 µm
with a Leica microtome (Leica RM2155, Nussloch, Ger-
many) and stained with haematoxylin and eosin (H&E) for
histological studies related to the infammatory process. The
histological sections were analysed using a Zeiss Axioscope
A1 optical microscope (Zeiss, Germany).
after adding PLA2. The enzymatic activity was performed
to observe the diference between the initial absorbance and
fnal absorbance and the results were expressed in percent-
age of enzymatic activity and half-maximal inhibitory con-
centration (IC50).
Computational analyses
A model of PLA2 from B. moojeni (UniProtKB P0C8M1)
was built by homology modelling using a toxin from Both-
rops jararacussu (PDBID 3JR8; dos Santos et al. 2011) as
a template. The model structure and stereochemistry were
evaluated with SwissModel (Waterhouse et al. 2018), and
the protonation state of every residue was determined with
Propka (Olsson et al. 2011). Calcium ions were added in
both monomers. Ligands LQFM011(5), LQFM043(6) and
LQFM044(7) were built with Avogadro (Hanwell et al.
2012) and minimised with Gaussian (Frisch et al. 2016). For
LQFM043 (6), two distinct atropisomers were generated—R
and S, LQFM043R(6a) and LQFM043S(6b), each with the
angle of 68.2º between the A and B rings (Fig. 3).
For immunohistochemistry, the sections were deparafn-
ised and rehydrated through a series of decreasing ethanol
concentrations. Antigen retrieval was performed in a citrate
bufer (pH 6.0). For the detection of primary antibodies,
the Leica Biosystems NovoLink Polymer Detection System
(RE7150-K, United Kingdom) kit was used. Endogenous
peroxidase activity was neutralised with Novocastra Per-
oxidase Block (3% hydrogen peroxide with stabilisers), and
Novocastra Protein Block (0.4% casein in PBS with stabilis-
ers) was subsequently used to reduce the nonspecifc bind-
ing of the primary antibody and polymer. Antibody against
TNF-α (rabbit polyclonal IgG, IM-0406, Imuny Biotechnol-
ogy, Brazil) was employed at a dilution of 1:50 incubated
overnight at 4 °C. Negative controls were obtained by omit-
ting the primary antibody incubation step. On the follow-
ing day, Novocastra Post Primary (rabbit anti mouse IgG)
and Novolink™ Polymer (anti-rabbit Poly-HRP-IgG) were
used as secondary antibodies. The sections were stained
with 3,3′-diaminobenzidinem (DAB) chromogen (1.74% w/v
DAB in a stabiliser solution) and NovolinkTM DAB Sub-
strate Bufer (polymer: bufered solution containing≤0.01%
hydrogen peroxide and preservative) (proportion 1:20). His-
tological sections were then analysed and digitised at the
same microscope light intensity (40×objective magnifca-
tion; six photomicrography felds/animal, n = 5 animals/
group; Axioscope A1 light microscope, Zeiss, Germany).
Measurement of the TNF-α-positive areas was performed
using Fiji software (Image J). The values obtained corre-
spond to the area positive for the TNF-α reaction (µm2) in
relation to the total area of the photomicrography feld. To
obtain the photomicrographs, the histological sections were
later counterstained by hematoxylin.
For molecular docking, DockThor (Custódio et al. 2014;
de Magalhães et al. 2014) in a standard docking mode was
used. Interactions between ligand and key residues were
detected and represented in two dimensions (2D) by Pose-
view (Stierand et al. 2010) and PLIP (Salentin et al. 2015).
Images were generated with the PyMol molecular graphics
system version 2.0 (Schrödinger) and InkScape (Harrington
et al. 2005).
Toxicity test
In vitro assay toestimate median lethal dose (LD50)
A neutral red uptake assay was performed according to
the standard protocol of Borenfreund and Puerner (1985),
modifed by ICCVAM (2006). The 3T3 fbroblast cell line
was cultured in DMEM supplemented with 10% (v/v) FBS,
100 IU/mL penicillin and 100 mg/mL streptomycin. The
cell cultures were maintained in ideal culture conditions and
were disaggregated from the culture fasks with confuence
between 80 and 90% using trypsin (0.025%)/EDTA (0.02%)
solution. Cell viability was previously determined using a
TC20™ automated cell counter (Hercules, CA, USA) fol-
lowing the manufacturer’s instructions. Viability values
greater than 90% were considered satisfactory for carrying
out the tests.
PLA2 activity
PLA2 activity was evaluated according to the method
described by Shiomi et al. (1998). First, 1.4 mL of 2% egg
yolk solution in 0.1 M Tris HCl (pH 8.0) with 50 µL of
LQM011(5), LQFM043(6) and LQFM044(7) at concentra-
tions of 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 or 25.6 µM/mL or saline
(white) was incubated with 50 µL of PLA2 from Bothrops
moojeni. The absorbance was monitored at 450 nm for 5 min
Thus, 3T3 fbroblasts (3× 103 cells/mL) were plated in
96-well plates 24 h prior to exposure to each compound.
Subsequently, compound LQFM011(5) was exposed to
eight concentrations in the range of 10,000 to 78.10 μM
(prepared in serial dilution). For compounds LQFM043(6)
1 3

Design, synthesis and pharmacological assessment of new pyrazole compounds
and LQFM044(7), the exposure concentrations were 1000
to 7.81 μM. After 48 h of exposure, the cells were incubated
with 250 μL per well for 3 h with 1% neutral red and 5%
FBS solution diluted in DMEM. The cells were subsequently
washed with PBS and incubated for 20 min with 150 μL of
an aqueous solution of ethanol (50%) and acetic acid (1%) on
a microplate shaker to ensure a homogeneous solution. The
absorption of the wells content was measured at 540 nm in
a spectrophotometer plate reader. The results are expressed
as mean standard error of the mean (SEM) of six repli-
cates in two independent experiments, expressed as a per-
centage by comparing the untreated control to the exposed
cells. IC50 values were determined using non-linear regres-
sion. The analysis was performed using GraphPad Prism 5.0
software (San Diego, CA, USA).
respectively, under Duf’s conditions (de Oliveira et al.
2013). The synthesis of 4-carbonitrile (17), (18) or (19) was
carried out through the formation of the oxime by reaction
of aldehydes (14), (15) or (16), respectively, with hydroxy-
lamine. This step was followed by in situ dehydration in the
presence of sodium iodide and DMF at a temperature of
120ºC for 12 h to aford (17), (18) and (19) with 54, 48 and
84% yield, respectively (Ballini et al. 2003). LQFM011(5),
LQFM043(6) and LQFM044(7) were synthetised through a
1,3-bipolar cycloaddition between 4-carbonitrile (17), (18)
or (19) and NaN3 using NH4Cl as the catalyst in DMF at
a temperature of 120ºC for 96 h to aford (5), (6) and (7)
with a 76, 85 and 96% yield, respectively (Zwaagstra et al.
1997). LQFM011(5), LQFM043(6) and LQFM044(7) were
obtained with an overall 29, 34 and 66% yield, respectively.
The estimated LD50 (dose that causes death in 50% of
animals) was calculated using the following equation:
Antinociceptive activity
log LD50(mmol∕kg) = 0.439 log IC50(mM) + 0.621.
Acetic acid‑induced abdominal writhing test
The LD50 values estimated for LQFM011(5),
LQFM043(6) and LQFM044(7) were used to classify the
probable toxicity of the compounds according to the United
Nations Globally Harmonized System of Classifcation and
Labelling of Chemicals (GHS).
Oral treatment with LQFM011(5), LQFM043(6) and
LQFM044(7) at doses of 70, 140 or 280 µmol/kg reduced
the number of writhings induced by acetic acid during
30 min of observation when compared to the control group
(group treated with vehicle). Indomethacin, the positive con-
trol, also reduced the number of writhings. There were sig-
nifcant diferences between LQFM043(6) and LQFM044(7)
compared to LQFM011(5) (Table 1).
Statistical analysis
The data were analysed statistically by one-way analysis of
variance (ANOVA) followed by the Newman–Keuls post
hoc test or a two-way ANOVA followed by the Bonfer-
roni post hoc test as a post-hoc. The statistical analysis was
carried out using GraphPad Prism version 5.0 software.
P<0.05 was considered signifcant.
Formalin test
In the early phase (0–5 min) of formalin-induced nocicep-
tion in mice, there was a signifcant diference between the
control (vehicle-treated) group, as well a signifcant reduc-
tion in response to the noxious stimulus during the late phase
(15–30 min) at 280 µmol/kg. Morphine produced a signif-
cant reduction in both phases and indomethacin (positive
control) showed a reduction only during the late phase, as
expected (Table 2).
Results
Synthesis
Synthesis of LQFM011( 5 ), LQFM043( 6 ) and LQFM044( 7 )
Randall–Selitto paw pressure test (mechanical
hyperalgesia)
As illustrated in Fig. 1b, the synthetic route to produce
5-(1-phenyl-1H-pyrazol-4-yl)-1H-tetrazole (5), 5-(1-(2-(tri-
fluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-tetrazole (6)
and 5-(1-(4-(trifuoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-
tetrazole (7) began through the classical method described
by Finar and Godfrey (1954). It used phenylhydrazines
(8), (9) or (10) with tetraethoxypropane to produce inter-
mediates (11), (12) and (13) with 84, 88 and 91% yield,
respectively. In turn, intermediates (11), (12) and (13)
were chemoselective and regiospecifcally formylated for
aldehydes (14), (15) and (16) with 83, 95 and 90% yield,
When evaluating mechanical hyperalgesia using the Ran-
dall–Selitto test, there were signifcant diferences in the
nociceptive threshold between treatment with LQFM011(5),
LQFM043(6) or LQFM044(7) compared to the control
(vehicle) at all time points during the test. When com-
paring activity among the compounds, there were signif-
cant differences in the first hour between LQFM044(7)
and LQFM011(5), as well as between LQFM043(6) and
LQFM044(7) for all time points (Table 3).
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J. C. Oliveria et al.
Fig. 1 a Structural design of LQFM011(5), LQFM043(6) and LQFM044(7) from, LQFM020(3) and LQFM039(4) lead compounds. b Synthetic
route for the preparation of LQFM011(5), LQFM043(6) and LQFM044(7) with all ligands
did not reduce the total number of leucocytes. There was a
reduction in polymorphonuclear cells for all compounds and
an increased number mononuclear cells for all compounds
compared to the control group (Table 5).
Anti‑infammatory activity
Carrageenan‑induced oedema
MPO activity was evaluated in the pleural exudate; all
three compounds and the positive control, dexamethasone,
signifcantly reduced this activity compared to the control
group. The levels of TNF-α were reduced by oral treat-
ment (280 µmol/kg) with LQFM011(5), LQFM043(6) and
LQFM044(7) or with dexamethasone compared to the vehi-
cle-treated group (Table 6).
Oral treatment with LQFM011(5), LQFM043(6) or
LQFM044(7) at a dose of 280 µmol/kg reduced paw oedema
at all time points of the test. The positive control (dexa-
methasone, 3.8 µmol/kg p.o.) also reduced paw oedema at all
time points (Table 4). When comparing the activity between
the compounds, there were signifcant diferences between
LQFM044(7) and LQFM011(5) for the fourth hour, as well
as between LQFM043(6) by LQFM044(7) for the second
and third hours.
Histology and quantifcation of TNF‑α
immunoreactivity
Carrageenan‑induced pleurisy
Morphological analysis showed that carrageenan admin-
istration caused an intense infammatory infltrate in the
dermis of the paws. In mice treated with LQFM011(5),
LQFM043(6), LQFM044(7) or dexamethasone, there was
a marked decrease in the migration of infammatory cells
In the test of carrageenan-induced pleurisy, oral treatment
with 280 µmol/kg of LQFM043(6) or LQFM044(7) or
3.8 µmol/kg of dexamethasone reduced the total number
of leucocytes compared to the control group. LQFM011(5)
1 3

Design, synthesis and pharmacological assessment of new pyrazole compounds
Table 1 Efect of LQFM011(5), LQFM043(6) and LQFM044(7) on
the number of acetic acid induced writhing in mice (n=8). Indometh-
acin was used as positive control
PLA2 inhibition and docking
LQFM011(5), LQFM043(6) and LQFM044(7) inhibited
PLA2 hydrolysis of phospholipids (Table 7). A model for B.
moojeni PLA2 (Fig. 4a) was obtained by homology model-
ling using the toxin from B. jararacussu as a template (which
shares 67.5% identity). The PLA2 catalytic site includes the
residues His48 and Asp49; it coordinates a calcium ion that
is necessary for the hydrolysis of lipids. Several related spe-
cies have an Asp49Lys mutation that hinders calcium coor-
dination, impeding PLA2 catalytic activity while toxic activ-
ity remains. Given that these residues are in a cleft, blocking
access is one way to inhibit PLA2 (Kang et al. 2011). Due to
its multiple actions in a host, we considered that the PLA2
catalytic site was responsible for its infammatory activity,
because the lipid catabolism appears to provoke an infam-
matory response. Therefore, the catalytic residues as the tar-
gets for the synthesised LQFMs (5–7) were used (Table 8).
The DockThor model was used to dock LQFM011(5),
LQFM043R(6a), LQFM043S(6b) and LQFM044(7). All
ligands formed hydrogen bonds with Lys109 from chain A
and Lys60 from chain B (Fig. 4b). This meant that all were
located close to the dimeric interface and possibly disturbed
the formation of the quaternary structure, which is relevant
for this activity (Fernandes et al. 2015). LQFM044(7) also
formed hydrogen bonds with Lys109A and Lys60B, as well
as a halogen bond between its trifuoromethyl group and
Tyr21B. LQFM043(6) showed diferences between the two
atropisomers, with LQFM043S(6b) having fewer interac-
tions compared to LQFM043R(6a). In addition, the latter
formed a halogen bond with Lys109A, a hydrogen bond with
Lys60B and hydrophobic interactions with Tyr27B, a key
residue for calcium coordination (Fernandes et al. 2015),
which is essential for catalytic activity.
Dose (µmol/kg No. writhing
p.o.)
% reduction
Vehicle
–
70
80.75 2.66
57.43 4.85b
61.29 4.64a
59.86 6.33a
54.88 2.41c
40.75 1,82c#
31.08 2.44c$
55.36 4.18c
36.68 3.57c$
32.52 2.62c$
37.58 3.74c
28.9
24.1
25.9
32.0
49.5
61.5
31.4
54.6
59.7
53,5
LQFM011
140
280
70
140
280
70
140
280
28
LQFM043
LQFM044
Indometacin
Percentage of reduction by number of writhings was calculated
a
b
based on the vehicle treated group values. P<0.05, P<0.01 and
cP<0.001 (compared with control group) according to ANOVA fol-
lowed by post-hoc Bonferroni’s test
#
Comparative diference with LQFM011 group values: P<0.01 and
$P<0.001 according to ANOVA followed by post-hoc Bonferroni’s
test
(Fig. 2). Although diapedesis was decreased, all groups had
oedematous lymphatic vessels.
TNF-α immunoreactivity was reduced in mice treated with
LQFM011(5), LQFM043(6) or LQFM044(7) (280 µmol/
kg) or dexamethasone (3.8 µmol/kg), to 6.857 444.1
µm2 (38.45% reduction, P < 0.05), 7.056 404.0 µm2
(36.67% reduction, P<0.001), 6.389 419.9 µm2 (42.65%,
P < 0.001) and 6.271 263.6 µm2 (43.71% reduction,
P < 0.001), respectively, compared to the vehicle treated
group (11.142 657.8 µm2) (Fig. 3).
Table 2 Efect of LQFM011(5),
Dose (µmol/kg, p.o.)
1º phase (pain
reaction time)
% reduction
2º phase (pain
reaction time)
% reduction
LQFM043(6) and
LQFM044(7), indomethacin
and morphine on the licking
time of the formalin test, in
mice (n=8)
Vehicle
–
62.5 3.9 s
–
34.0
45.0
13.6
–
193.9 5.93 s
97.63 8.0 s
111.45 5.14 s
109.06 5.4 s
79.35 10.5 s
2.6 1.0 sc
–
LQFM011
LQFM043
LQFM044
Indometacin
Morphine
280
280
280
28
41.25 4.5 sa
34.34 3.12 sb
39.7 5.31 sa
54.0 2.53 s
2.5 0.7 sc
49.6
19.1
39.1
59.1
98.66
7.4 (s.c.)
96.0
Early phase, 0–5 min; late phase, 15–30 min
Pain reaction time was calculated based on the vehicle treated group values of each phase, in seconds
Early phase (1º phase), 0–5 min; late phase (2º phase), 15–30 min
%: percentage of reactivity reduction
aP<0.05, bP<0.01 and cP<0.001 (compared with control group) according to ANOVA followed by Bon-
ferroni’s test
1 3

J. C. Oliveria et al.
Table 3 Efect of LQFM011(5), LQFM043(6) and LQFM044(7) and dexamethasone (positive control) on the mechanic hyperalgesia test, in
mice (n=8) induced by carrageenan
Dose
1st hour
% reduction 2nd hour
% reduction 3rd hour
% reduction 4th hour
% reduction
(µmol/kg,
p.o.)
Vehicle
–
168 2.5
–
168 4.66
127 3.1c
138 4.8 c
162 3.5
140 2.1
LQFM011 280
LQFM043 280
LQFM044 280
145.2 4.5c 13.6
24.4
17.8
122.5 4.6 c 24.4
102.7 3.4 c 26.6
152 3.2 a
124 5.4 c$* 26.2
118 6.6 c
30
9.5
133 3.7 c
111 4.0 c*
98 4.0 c
17.9
31.5
39.5
113 3.3 c
98 3,9 c”
92.2 3.2 c
19.3
30.0
34.1
113 3.9 c* 32.7
95.5 4.4 c 43.1
Dexa
3.8
a
c
The diference of withdrawal threshold was calculated based on the vehicle treated group values. P<0.05, P<0.001 (compared with control
group) according to two-way ANOVA followed by Bonferroni’s post-hoc test. Comparative diference based on the LQFM011 group treated val-
ues: $P<0.001 according to ANOVA followed by post-hoc Bonferroni’s test
*
Comparative diference with LQFM043 group treated values: P<0.05 and P<0.001 according to ANOVA followed by post-hoc Bonferroni’s
test
Dexa dexamethasone; %: percentage of threshold reduction
Table 4 Efect of LQFM011(5), LQFM043(6) and LQFM044(7) or dexamethasone (positive-control) on the carrageenan-induced edema test, in
mice (n=8)
Dose (µmol/kg) 1st hour
% reduction 2nd hour
% reduction 3rd hour
102.2 6.2
% reduction 4th hour
% reduction
Vehicle
–
107.8 7.4
–
91.2 6.3 15.6
88.0 6.3 a 18.4
78.9 6.5 c 26.8
71.1 5.6 c 34.0
116.7 8.6
–
–
92.2 4.3
75.7 3.8
–
17.9
LQFM011 280
LQFM043 280
LQFM044 280
84.2 6.1c 28.0
94.4 3.4b 19.1
71.1 3.9c£ 39.1
61.1 5.1c 47.6
76.6 3.8b 25.0
81.0 4.1a 20.7
61.1 4.5c” 40.2
51.1 3.5c 50.0
73.0 4.5a 20.8
57.8 4.0c¢ 37.3
46.6 3.7c 49.4
Dexa
3.8
a
b
c
The values were expressed as mean SEM of the diference between the paws, in µL P<0.05, P<0.001, P<0.001, compared to control
group, according to two-way ANOVA followed by Bonferroni’s post-hoc test
Comparative diference with LQFM011 group treated values: ¢P<0.05 and $P<0.001 according to ANOVA followed by post-hoc Bonferroni’s
test
”
£
Comparative diference with LQFM043 group treated values: P<0.05 and P<0.01 according to ANOVA followed by post-hoc Bonferroni’s
test
Values represent the means SEM (n=8) of diference of between the paws in µL
Dexa dexamethasone; %: percentage of reduction paw edema
Toxicity test
Discussion
OECD Guidance Document No. 129 (2012) prescribes that
the IC50 value found after exposure of the test substance
to 3T3 basal fbroblasts can be used to estimate the ini-
tial doses for acute toxicity. 3T3 cells were exposed to
LQFM011(5) (10,000 to 78.10 μM); the IC50 was 630 μg/
mL. For the evaluation of compounds LQM043(6) and
LQFM044(7), the IC50 values were 204.6 μg/mL and
60.6 μg/mL, respectively (Table 8). Based on the IC50
values, the LD50 values were predicted as 1162.39 mg/
kg, 764.99 mg/kg and 486.49 mg/kg for LQFM011(5),
LQFM043(6) and LFM044(7), respectively, placing all
compounds in category 4 based on the GHS.
To pharmacologically evaluate compounds (5–7), we frst
examined anti-nociceptive activity; the writhing abdomi-
nal test was performed with three doses. This is a general,
nonspecifc model and does not distinguish an antinocicep-
tive from an anti-infammatory response (Bars et al. 2001).
The dose (280 µmol/kg) was chosen for the subsequent tests
because there were diferent response profles in the animals
treated with the compounds (5–7). The action of the com-
pounds in two types of pain, neurological and infammatory,
was evaluated by the formalin test. The results reinforced
the presence of antinociceptive activity and suggest that the
compounds modulate pain mediators, such as histamine,
1 3

Design, synthesis and pharmacological assessment of new pyrazole compounds
Table 5 Efect of LQFM011(5), LQFM043(6) and LQFM044(7) or dexamethasone (positive control) in cell migration, in model carrageenan-
induced pleurisy in mice (n=8)
Dose
Total leuko-
cytes
% reduction Poly
% reduction Mono
% increase
(µmol/kg)
Vehicle
LQFM011
LQFM043
–
280
280
5.35 0.21
5.71 0.70c
–
–
3.70 0.18
–
1.64 0.18
–
2.29 0.46c 38.1
3.37 0.48c
3.46 0.09c
105.4
110.9
4.16 0.12c$ 22.2
3.51 0.13c$* 34.3
0.70 0.09
81.0
c #
LQFM044
Dexa
280
3.8
0.53 0.14
85.6
2.98 0.14b
1.85 0.18
81.7
–
c $
2.43 0.09c
54.5
0.58 0.18c 84.3
Percentage of reduction by number of total leukocytes, polymorphonuclear, and mononuclear was calculated based on the vehicle treated group
values. bP<0.01 and cP<0.001 (compared with control group) according to ANOVA followed by Bonferroni’s test
Comparative diference with LQFM011 group treated values: #P<0.01 and $P<0.001 according to ANOVA followed by post-hoc Bonferroni’s
test
Comparative diference with LQFM043 group treated values: *P<0.001 according to ANOVA followed by post-hoc Bonferroni’s test
Values represent the means SEM (n=8) of number of leukocytes/mL×106
Dexa dexamethasone, poly polymorphonuclear, mono mononuclear; % percentage of leukocytes reduction and increase by mono
Table 6 Efect of LQFM011(5),
Dose (µmol/kg)
Myeloperoxidase % reduction
(mU/mL)
TNF-α (pg/mL)
% reduction
LQFM043(6) and LQFM044(7)
or dexamethasone (positive
control) in the activity of
Vehicle
–
122.4 8.95
97.20 7.61a
86.19 15.93a
71.81 7.60b
56.39 6.17c
-
35.82 1.37
27,5 2.36a
21.23 1.30b
20.52 4.4c
5.89 0.9c
myeloperoxidase and level of
TNF-α, in model carrageenan-
induced pleurisy in mice (n=8)
LQFM011
LQFM043
LQFM044
Dexa
280
280
280
3.8
20.6
29.6
41.3
53.3
23.2
40.7
42.7
83.5
Percentage of activity reduction of myeloperoxidase (mU/mL) and reduction of TNF-α (pg/mL) was calcu-
lated based on the vehicle treated group values. aP<0.05, bP<0.01 and cP<0.001 (compared with control
group) according to ANOVA followed by Bonferroni’s test
Values represent the means SEM (n=8)
Dexa dexamethasone
serotonin and bradykinin during the frst phase (neurological
pain) and on cytokines (TNF-α) and PGE2 during the second
phase (infammatory pain). In other words, these compounds
participate in more than one mechanism (Shibata et al. 1989;
Bars et al. 2001). Oliveira et al. (2017) reported similar fnd-
ings using LQFM020(3) and LQFM039(4). The data dem-
onstrated a variable reduction in the nociceptive threshold
in the model of infammatory pain and Randall–Selitto test
at all times. These fndings reafrm the action regarding
mediators responsible for the sensitisation of nociceptors.
The anti-infammatory action of the synthesised com-
pounds was examined by the carrageenan-induced paw
oedema model. This test is used to evaluate anti-infamma-
tory activities of new compounds (Villar et al. 1987; Vinegar
et al. 1987). The data clearly demonstrated the presence of
infammatory activity, with reduced oedema at all times for
LQFM043(6) and LQFM044 (7), and from the second to
the fourth hour for LQFM011(5). The number of leucocytes
was counted in the infamed pleural exudate; all compounds
except LQFM011(5) reduced the total number of leucocytes.
However, there was a reduction in polymorphonuclear cells
and an increase in mononuclear cells. The reduced MPO
activity justifed the reduction in polymorphonuclear cells
(Aratani 2018; Khan et al. 2018), indicating a resolutive pro-
fle. There was reduced release of pro-infammatory TNF-α;
this fnding is informative because this cytokine can regulate
several cellular and biological processes, such as immune
function, cell diferentiation, proliferation and apoptosis
(Lawrence 2009; Liu et al. 2017). The histological analy-
ses demonstrated alterations in the infamed tissue of the
untreated animals (vehicle carrageenan), with the accumula-
tion of several infammatory cells, leading to polymorphonu-
clear cells and tissue disruption, with oedema. However, the
animals treated with compounds 5–7 presented a reduction
in polymorphonuclear cells and an increase in the migration
of mononuclear cells, and the immunostaining with anti-
TNF-α demonstrated the reduction of TNF-α. These data
are interesting and corroborate fndings in the pleurisy test.
1 3

J. C. Oliveria et al.
Fig. 2 Histopathology of the
paw dermis of the control
group not injured (a), control
group injured (b) and groups
treated with LQFM011(5)
(c), LQFM043(6) (d) and
LQFM044(7) (e) and dexa-
methasone (f). The absence
of cellular infltration was
observed in the group VEHI-
CLE (SALINE) (a), the group
VEHICLE (CARRAGEENAN)
showed prominent infamma-
tory cells infltration (b), while
LQFM011(5) (c), LQFM043(6)
(d), LQFM044(7) (e) and dexa-
methasone (f) were reduced for
this infltration. The photomi-
crographs were taken with×40
magnifcation, scale bar, 20 µm
(staining: hematoxylin–eosin),
infammatory cells (thick
arrows) and lymphatic vessels
(thin arrows)
All tested compounds inhibited PLA2 in vitro at high
concentrations. LQFM043(6) and LQFM044(7) presented a
lower IC50 compared with LQFM011(5). These results sug-
gest that PLA2 inhibition may be one of the mechanisms of
action for the synthesised compounds (5–7). To better under-
stand these intermolecular interactions, we performed dock-
ing studies with these compounds. The predicted intermolec-
ular interactions between LQFM043R(6a) and LQMF044(7)
indicate more interactions in the catalytic cleft. Hence, these
compounds occupy the available space and prevent normal
enzymatic activity.
was verifed, and acute oral toxicity was assessed as 4
according to GHS (OECD 2012).
The diferences found in the interactions with the PLA2
catalytic site are related to the presence of the CF3 allo-
gene, Specifcally, LQFM011(5), which contains CF3,
presented lower inhibition of the enzyme in the in vitro
test compared to the other synthesised compounds. These
diferences could indicate greater or lesser action in the
pharmacological tests, which was seen in most tests, such
as abdominal writhing, hyperalgesia, paw oedema, cell
migration and the TNF-α level in infamed tissue. The
presence of CF3 also interfered with the toxicity of the
molecule but did not change the classifcation of the com-
pounds LQFM043 (6) and LQFM044 (7) in relation to
LQFM011 (5) according to the GHS.
This fnding corroborates the PLA2 inhibition presented
in the in vitro test and the pharmacological test results
obtained in the abdominal writhing (at doses of 140 and
280 µmol/kg), hyperalgesia, paw oedema, cell migration
and TNF-α quantifcation. The toxicity of the compounds
1 3

Design, synthesis and pharmacological assessment of new pyrazole compounds
Fig. 3 Immunoreactivity for
TNF-α with immunostaining (a)
and quantifcation for TNF-α (b)
by hind paw of animals treated
with compounds LQFM011(5),
LQFM043(6), LQFM044(7)
and dexamethasone. A The
group VEHICLE (CARRAG
EENAN) showed prominent
infammatory cells infltra-
tion with marking for TNF-α
(a), while LQFM011(5) (c),
LQFM043(6) (d), LQFM044(7)
(e) and dexamethasone (f)
had reduced marking. Repre-
sentative photomicrographs
(×20 magnifcation, scale bar,
50 µm). The arrows indicate
the intensity of TNF measured
in the tissue. B Quantifcation
TNF-α, data are reported as
mean SEM for 5 diferent
animals (6 photomicrography
felds/animal). Calculated based
on the vehicle treated group
values. aP<0.05, bP<0.01,
cP<0.001 and $P<0.001
according to ANOVA one-way
followed by Newman–Keuls test
1 3

J. C. Oliveria et al.
Table 7 Inhibition of PLA2 by LQFM011(5), LQFM043(6) and
LQFM044(7)
Conclusion
The synthesised compounds modulate important mediators
to exert antinociceptive and anti-infammatory activity, with
a reduced pro-infammatory profle, prevalence of an anti-
infammatory profle and pharmacological safety. One of the
mechanism of action produced by these compounds is PLA2
inhibition, specifcally the interaction of compounds (5–7)
with crucial amino acid residues in the active site. Although
compound LQFM011(5) demonstrated antinociceptive and
anti-infammatory activities, the absence of the CF3 allogene
resulted in lower activity compared to other compounds.
Compound
Concentration Inhibition (%)
(µmol/mL)
IC50 (µmol/mL)
LQFM011
6.4
12.8
25.6
6.4
12.8
25.6
6.4
16.59
25.28
34.12
26.96
38.96
50.70
8.44
92.07
25.29
34.38
LQFM043
LQFM044
12.8
25.6
17.78
48.89
Fig. 4 Model of PLA2 and its interactions with LQFM011(5),
LQFM043(6) and LQFM044(7). a Overview of the model of PLA2
of Bothrops moojeni, showing both secondary structure and protein
surface. b Molecular interactions of PLA2, showing diferent interac-
tions depending on the atropoisomer submitted
1 3

Design, synthesis and pharmacological assessment of new pyrazole compounds
the role of nitric oxide. Nitric Oxide 30(69):35–44. https://doi.
org/10.1016/j.niox.2017.04.006
Table 8 LD50
values viability of 3T3 cells after exposure
to LQFM011(5) (10,000 to 78.10 μM) and LQMF043(6)
and LQFM044(7) (1000 to 7.81 μM) (C) for 48 h according
to OECD guideline 129
Florentino IF, Silva DPB, Cardoso CS, Menegatti R, Carvalho
FS, Lião LM, Pauo M, Pinto PM, Peigneur S, Costa EA, Tyi-
gat J (2019) Antinociceptive efects of new pyrazoles com-
pounds mediated by the ASIC1α channel, TRPV-1 and µMOR
receptors. Biomed Pharmacother 115(2):1–12. https://doi.
org/10.1016/j.biopha.2019.108915
LD50 (mg/kg)
GHS
classif-
cation
Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,
Cheeseman JR, Scalmani G, Barone V, Petersson GA, Nakat-
suji H, Li X, Caricato M, Marenich AV, Bloino J, Janesko, BG,
Gomperts R, Mennucci B, Hratchian HP, Ortiz JV, Izmaylov
AF, Sonnenberg JL, Williams-Young D, Ding F, Lipparini F,
Egidi F, Goings J, Peng B, Petrone A, Henderson T, Ranas-
inghe D, Zakrzewski VG, Gao J, Rega N, Zheng G, Liang W,
Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M,
Nakajima T, Honda Y, Kitao O, Nakai H, Vreven,T, Throssell
K, JAJr Montgomery, Peralta JE, Ogliaro F, Bearpark MJ, Heyd
JJ, Brothers EN, Kudin KN, Staroverov VN, Keith TA, Kob-
ayashi R, Normand J, Raghavachari K, Rendell AP, Burant JC,
Iyengar SS, Tomasi J, Cossi M, Millam JM, Klene M, Adamo
C, Cammi R, Ochterski JW, Martin RL, Morokuma K, Farkas
O, Foresman JB, Fox DJ (2016) Gaussian 16, revision C.01.
Gaussian, Wallingford
LQFM011
LQFM043
LQFM044
1162.39
764.99
486.49
4
4
4
The values are predicted doses and GHS classifcation
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