Mechanisms involved in the antinociceptive and anti-inflammatory effects of a new triazole derivative: 5-[1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl]-1H-tetrazole (LQFM-096)

Article abstract of DOI:10.1007/s10787-020-00685-8

The aim of this study was to design, synthesize and evaluate the potential analgesic and anti-inflammatory effects of 5-[1-(4-fluorphenyl)-1H-1,2,3-triazol-4-yl]-1H-tetrazole—(LQFM-096: a new triazole compound) as well as to elucidate its possible mechani

Full text of DOI:10.1007/s10787-020-00685-8

Infammopharmacology
https://doi.org/10.1007/s10787-020-00685-8
Inflammopharmacology
ORIGINAL ARTICLE
Mechanisms involved in the antinociceptive
and anti‑infammatory eꢀects oꢁ a new triazole derivative:
5‑[1‑(4‑fuorophenyl)‑1H‑1,2,3‑triazol‑4‑yl]‑1H‑tetrazole (LQFM‑096)
1
1
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1
1
Carina S. Cardoso · Daiany P. B. Silva · Dayane M. Silva · Iziara F. Florentino · James O. Fajemiroye ·
1
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Lorrane K. S. Moreira · José P. Vasconcelos · Germán Sanz · Boniek G. Vaz · Luciano M. Lião · Danilo da S. Lima ·
Fernanda Cristina A. dos Santos · Ricardo Menegatti · Elson A. Costa
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Received: 4 December 2019 / Accepted: 24 January 2020
©
Springer Nature Switzerland AG 2020
Abstract
The aim of this study was to design, synthesize and evaluate the potential analgesic and anti-inꢀammatory eꢁects of
-[1-(4-ꢀuorphenyl)-1H-1,2,3-triazol-4-yl]-1H-tetrazole—(LQFM-096: a new triazole compound) as well as to elucidate its
5
possible mechanisms of action. The oral administration of LQFM-096 (10, 20 or 40 mg/kg) decreased the number of writh-
ing in mice. At the dose of 20 mg/kg, LQFM-096 reduced the licking time at both neurogenic and inꢀammatory phases of
the formalin test. Pretreatment with naloxone (3 mg/kg) and glibenclamide (3 mg/kg) attenuated the antinociceptive eꢁect
of LQFM-096 in the ꢂrst phase of the formalin test. At the dose of 20 mg/kg, LQFM-096 also decreased the licking time
in the acidiꢂed saline-induced and capsaicin-induced nociception. This eꢁect was blocked by naloxone (3 mg/kg) pretreat-
ment prior to the administration of LQFM-096. In addition, LQFM-096 inhibited hyperalgesia induced by carrageenan and
PGE2. Naloxone (3 mg/kg) attenuated the eꢁect of LQFM-096 through disinhibition of PGE2-induced hyperalgesia. The
anti-inꢀammatory eꢁect of LQFM-096 was demonstrated in carrageenan-induced oedema or pleurisy as well as CFA-induced
arthritis. The hyperalgesia and cellular migration in CFA-induced arthritis were reduced signiꢂcantly. Altogether, these
ꢂndings suggest antinociceptive eꢁect of LQFM-096 and implicate the modulation of ASICs/TRPV1 channels by opioid/
KATP pathway. The anti-inꢀammatory eꢁect of LQFM-096 was mediated by a reduction in oedema, leukocytes migration,
TNF-α, PGE levels and myeloperoxidase activity.
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Keywords Pain · Inꢀammation · Opioid receptors · ASICs · TRPV1 · TNF-α
Introduction
Pain is an unpleasant sensory and emotional experience that
is associated with an actual or potential tissue injury (IASP
1994). It alerts the organism to external stimuli or threats
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10787-020-00685-8) contains
supplementary material, which is available to authorized users.
4
*
Elson A. Costa
xico@ufg.br
Chemistry Institute, Federal University of Goiás, Campus
Samambaia, Goiânia, GO, Brazil
5
Department of Histology, Embriology and Cell Biology,
1
Department of Pharmacology, ICB, Federal University
of Goiás, Campus Samambaia, 314, Goiânia, GO 74001-970,
Brazil
ICB, Federal University of Goiás, Campus Samambaia, 314,
Goiânia, GO 74001-970, Brazil
6
Laboratório de Farmacologia de Produtos Naturais e
2
3
Faculty of Pharmacy, Laboratory of Medicinal
Pharmaceutical Chemistry, Federal University of Goiás,
Goiânia, GO, Brazil
Sintéticos – ICB-2, Universidade Federal de Goiás, Sala 216,
Goiânia, GO CEP 74001-970, Brazil
Laboratory of Chromatography and Mass Spectrometry,
Chemistry Institute, Federal University of Goiás, Goiânia,
GO, Brazil
Vol.:(0
1
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C. S. Cardoso et al.
and triggers appropriate protective responses (Fein 2011).
The noxious stimuli often activate nociceptor ꢂbres to pro-
mote signal transduction and transmission to the brain (Gold
and Gebhart 2010; Julius and Basbaum 2001).
RING BIOISOSTERISM
The transient receptor potential vanilloid 1 (TRPV1)
channel and acid-sensing ion channels (ASICs) play impor-
tant roles in modulating nociceptive signalling. The inꢀux of
N
C
N
C
N
N
N
N
N
N
+
2+
H
N
N
H
Na and Ca through these channels can trigger membrane
depolarization and signal transduction cascades (Deval et al.
B
B
N
N
N
2
010; Deval and Lingueglia 2015; Wemmie et al. 2013).
Pain development often involves inꢀammatory mediators
A
A
such as prostaglandins, bradykinin, cytokines, glutamate,
substance P, and calcitonin gene-related peptide. These
chemical mediators often sensitize and activate peripheral
nociceptors to induce hyperalgesia and allodynia (Ji et al.
F
F
LQFM-020 (1)
LQFM-096 (2)
2
014; Oliveira-Junior et al. 2016).
Fig. 1 Structural design of LQFM-096 (2) from LQFM-020 (1) lead
Opioids and nonsteroidal anti-inflammatory drugs
compound
(
NSAIDs) are the leading analgesic drugs for the treat-
ment of pain. However, these drugs are associated with side
eꢁects such as gastrointestinal, sedation, dependence, and
tolerance (Benyamin et al. 2008; Jage 2005; Porreca and
Ossipov 2009). Hence, a search for eꢁective and safer anal-
gesics has become a necessity.
Materials and methods
Chemistry
Triazole compounds are known for their antibacterial,
antifungal, anticancer, and neuroprotective activities (Gupta
and Jain 2017; Penthala et al. 2015; Li et al. 2016). This
pharmacological proꢂle is associated with the broad interest
in medicinal chemistry of triazole compounds and therapeu-
tic applications.
General
The reactions were monitored by TLC using commer-
cially available pre-coated plates (Whatman 60 F254
silica). The developed plates were examined under UV
1
13
light (254 and 365 nm). The H and C-NMR spectra
were recorded in the indicated solvent on a Bruker Avance
III 500 MHz spectrometer. Chemical shifts were quoted
in parts per million downꢂeld of TMS, and the coupling
constants were expressed in Hertz. All assignments of the
Although some studies have shown anti-inꢀammatory
and antinociceptive activities of triazole derivatives (Huna-
shal et al. 2014; Joanna et al. 2014; Khanage et al. 2013),
the underlying mechanisms of these actions remain unclear.
The present study sought to synthesize and investigate the
biological activities of LQFM-096 as well as elucidate its
possible mechanisms of action.
1
13
signals of H and C NMR spectra are consistent with the
chemical structures of the products. Infrared spectra were
recorded on a Perkin-Elmer Spectrum Bx-II FT-IR Sys-
tem spectrophotometer instrument as ꢂlms on KBr discs.
Melting points were assessed using a Marte melting point
apparatus, and the results were uncorrected. The organic
solutions were dried over anhydrous sodium sulphate. The
organic solvents were removed under reduced pressure in a
rotary evaporator. Mass spectra (MS) were obtained with
a microTOF III (Brucker Daltonics Bremen, Germany).
To prepare a sample for MS analysis, 1 µg of the sample
was diluted in 1 ml of methanol. To perform the analy-
sis in positive mode, 1 μl of formic acid was added to
the sample and the resulting solution was infused into the
ESI source at a ꢀow rate of 3 μl/min. The ESI(+) source
conditions were as follows: nebulizer with nitrogen gas;
pressure, 0.4 bar; temperature, 200 °C; capillary voltage,
− 4 kV, transfer capillary temperature, 200 °C; drying
gassed, 4 l/min; end plate oꢁset, − 500 V; skimmer, 35 V;
The LQFM-096 was designed through ring bioisoster-
ism strategy, where pyrazole scaꢁold present in LQFM-020
(
Oliveira et al. 2016) was changed by 1,2,3-triazole scaꢁold
present in LQFM-096 (Fig. 1). The bioisoterism strategy is
commonly used in medicinal chemistry drug design (Lima
and Barreiro 2005). There are many drugs which showed
the 1,2,3-triazole scaꢁold, i.e. cefatriazine, ruꢂnamide, and
tazobactam, as well as, in many lead compounds (Dheer
et al. 2017). Once this scaꢁold is present on many drugs and
lead compounds, it is considered as a privileged structure, in
drug designed (Yet 2018). The ꢀuoride scaꢁold was main-
tained in order to facilitate solubility in water and biological
ꢀ
uids (Hagmann 2008; Wang et al. 2014). Besides that, the
diꢁerence in the physical–chemical properties due to scaf-
fold changes between LQFM-020 and LQFM-096 results in
expectations of change in the new compound eꢁectiveness
in the used experimental models.
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Mechanisms involved in the antinociceptive and anti‑infammatory eꢀects oꢁ a new triazole…
and collision voltage, −1.5 V. Each spectrum was acquired
using 2 microscans. The resolving power is m/Δm50%
(δ): 8.99 (s, H-5), 8.06 (dd, 8.8; 4.7,H-2′), 8.06 (dd, 8.8; 4.7,
H-6′), 7.50 (dd, 8.8; 8.4, H-3′), 7.50 (dd, 8.8; 8.4, H-5′); 2D
1
6,500.00, where Δm50% is the peak full width at the
NMR (HSQC/HMBC-125.76 MHz) CDCl /TMS (δ): 162.2
3
half-maximum peak height. Mass spectra were acquired
and processed with data analysis software (Brucker Dal-
tonics, Bremen, Germany). Microwave reactions were con-
ducted using a CEM Discovery microwave reactor.
(C-4′), 142.4 (C-5′’), 133.5 (C-1′), 122.9 (C-2′ and 6′), 121.2
+
(C-4), 120.8 (C-5), 117.3 (C-3′ and 5′); [M + H] m/z of
232.0708; purity>98% (supporting information).
Pharmacology
Synthesis oꢁ 1‑(4‑fuorophenyl)‑1H‑1,2,3‑triazole‑4‑carbon‑
itrile (6) (Portman 1998)
Animals
Distilled water (20 ml) was added to a heated heterogeneous
mixture of 1-azido-4-ꢀuorobenzene (4) (548 mg, 4 mmol)
and 2-chloroacrylonitrile (5) (696 mg, 8.00 mmol), and
the mixture was heated at 80 °C for 24 h. At the end of
the reaction, excess 2-chloroacrylonitrile (5) was removed
through distillation, cyclohexene (5 ml) was added, and the
suspension was stirred for 20 min. The precipitate was ꢂl-
tered under vacuum and dried. The phases were separated
and the aqueous layer was extracted with CH Cl . The
The experiments were performed using female Swiss albino
mice (25–30 g; 7 weeks old) from the Central Animal House
of the Federal University of Goiás (UFG). Animals were
maintained under controlled temperature (22 ± 2 °C) and
luminosity (12 h light/dark cycle) with free access to pellet
food and water. The animals were acclimatized for 7 days
before beginning the experiments. All of the in vivo stud-
ies were previously approved by the Ethics Committee in
Animal Experimentation of the Federal University of Goiás
(CEUA/UFG, Date of Approval 07/08/2013; Protocol:
017/13). As recommended by the ethics committee, at the
end of all the acute tests, the animals were euthanized by
cervical dislocation. At the end of the CFA-induced arthritis,
the animals were euthanized by decapitation. To prevent bias
in the research results, the experimenters were unaware of
which animal was receiving a particular treatment.
2
2
combined organic layers were dried (Na SO ) and con-
2
4
centrated in a vacuum, and the crude product was puriꢂed
by column chromatography (SiO , CH Cl /MeOH=98:2)
2
2
2
to 1-(4-ꢀuorophenyl)-1H-1,2,3-triazole-4-carbonitrile (6)
(
661 mg, 88%) as a beige solid with m.p. =122–124 °C and
−
1
Rf = 0.60 (hexene/AcOEt = 7:3). IR
(KBr) cm : 3132
max
(
ν C-H), 2249 (ν CN), 1515(ν C=C), and 835 (ν Ar-1,4);
1
H-NMR (500.13 MHz) CDCl (δ): 8.43 (s, H-5), 7.74 (dd,
3
9
.1; 4.5, H-2′), 7.74 (dd, 9.1; 4.5, H-6′), 7.30 (dd, 9.1; 7.8,
H-3′), 7.30 (dd, 9.1; 7.8, H-5′); 2D NMR (HSQC/HMBC-
25.76 MHz) CDCl /TMS (δ): 163.3 (d, 3.7, C-4′), 132.0
Drugs and chemicals
1
LQFM-096 was synthesized at the Laboratory of Medici-
nal Pharmaceutical Chemistry, Faculty of Pharmacy, UFG
as described above. The following drugs were purchased
through a pharmaceutical company representative: acetic
acid (Synth, Brazil); dimethyl sulphoxide (DMSO) (Sigma
Chemical, USA), amiloride (Sigma Chemical, USA), capsai-
cin (Sigma Chemical, USA), capsazepine (Sigma Chemical,
USA), carrageenan (Sigma Chemical, USA), formaldehyde
3
(
d, 3.7, C-1′), 127.6 (s, C-5), 123.2 (d, 8.7, C-2′ and 6′),
1
22.2 (s, C-4), 117.3 (d, 23.3, C-3′ and 5′), 111.0 (s, C-6);
+
[
M+H] m/z of 189.0543.
Synthesis oꢁ 5‑(1‑(4‑fuorophenyl)‑1H‑1,2,3‑tria‑
zol‑4‑yl)‑1H‑tetrazole—(LQFM 096) (2) (Alterman and Hall‑
berg 2000; Zwaagstra et al. 1997)
(
Synth, Brazil), indomethacin (Prodome, Campinas, SP,
®
Sodium azide (780 mg, 12 mmol) and ammonium chloride
Brasil), morphine sulphate (Dimorf , Cristalia, SP, Bra-
®
(
642 mg, 12 mmol) in 10 ml of DMF were added to a heated
zil), naloxone hydrochloride (Narcan ), and glibenclamide
heterogeneous mixture of 1-(4-ꢀuorophenyl)-1H-1,2,3-tri-
azole-4-carbonitrile (6) (188 mg, 1 mmol). The resulting
mixture was reacted by microwave irradiation (20 W) at
(Sigma-Aldrich, St. Louis, MO, USA). LQFM-096 was dis-
solved in 10% DMSO in saline, and all other drugs were
dissolved in 0.9% saline.
1
00 °C for 20 min and 1 min of ramp time. The reaction
mixture was poured out and acidiꢂed to pH=5, and the pre-
cipitate formed was ꢂltered oꢁ under vacuum and dried.
The crude product was crystallized from (DMF/AcOH=9:1)
to 5-(1-(4-ꢀuorophenyl)-1H-1,2,3-triazol-4-yl)-1H-tetra-
zole—(LQFM-096) (2) (203 mg, 88%) as a beige solid with
m.p. =223–226 °C and Rf=0.58 (CH Cl /MeOH=95:5).
Antinociceptive activity
Acetic acid‑induced abdominal writhing Acetic acid-
induced abdominal writhing was performed as described by
Koster et al. (1959). The animal groups (n=8) were treated
orally (p.o.) with vehicle (10% DMSO, 10 ml/kg), LQFM-
096 (10, 20, or 40 mg/kg) or indomethacin (10 mg/kg, posi-
tive control) prior to the administration of the acetic acid
2
2
−1
IR (KBr) cm : 3500–3050 (ν N–H), 3029 (ν C-H), 1660
max
1
(
ν C=C), and 841 (ν Ar-1,4); H-NMR (500.13 MHz) CDCl
3
1
3

C. S. Cardoso et al.
solution (1.2% v/v; 10 ml/kg, intraperitoneally [i.p.]). Each
mouse was placed in a big glass cylinder and the number of
writhing occurrences was recoded over a 30 min period after
the administration of acetic acid. The results were expressed
as the mean± S.E.M of the number of writhing.
results were expressed as the mean± S.E.M of the licking
time (s).
The involvement of the opioid system in the effect
of LQFM-096 on PGE -induced hyperalgesia was also
2
investigated. The experimental protocol was performed as
described in section “Antihyperalgesic activity”. The experi-
mental groups of mice (n=10) were pretreated with nalox-
one (3 mg/kg, i.p.), nonselective opioid receptor antagonist
or saline (10 ml/kg) ꢂfteen min prior to oral treatment with
the vehicle (10 ml/kg, 10% DMSO), LQFM-096 (20 mg/kg)
or morphine (5 mg/kg, s.c.). The results were expressed as
the mean± S.E.M of the diꢁerence between the nociceptive
thresholds of the paws (g).
Formalin‑induced nociception Formalin-induced nocic-
eption was conducted as described by Hanskaar and Hole
(
1987). The experimental groups of mice (n=8) were treated
with vehicle (10% DMSO, 10 ml/kg, p.o.), LQFM-096
20 mg/kg, p.o.), or indomethacin (10 mg/kg, p.o.—positive
(
control in the second phase) or subcutaneously (s.c.) with
morphine (5 mg/kg, positive control in the ꢂrst phase where
direct stimulation of the nociceptors causes neurogenic pain
and in the second phase where the release of inꢀammatory
mediators causes inꢀammatory pain). After an interval of
sixty min, 20 μl of 3% formalin was injected into the plantar
surface of the right hind paw prior to the placement of an
individual mouse in an acrylic box. A mirror was placed
under the box to ensure unhindered observation of the for-
malin-injected paw for 30 min. Pain reaction time (licking
time) was assessed during the following two periods: from 0
to 5 min (ꢂrst phase) and from 15 to 30 min (second phase).
The results were expressed as the mean± S.E.M of the lick-
ing time in seconds (s).
ASIC Involvement To evaluate the involvement of ASICs
in the antinociceptive eꢁects of LQFM-096, mice (n= 8)
were treated with vehicle (10 ml/kg, 10% DMSO, p.o.),
LQFM-096 (20 mg/kg, p.o.), amiloride (10 mg/kg, i.p.,
ASIC inhibitor), or morphine (5 mg/kg, s.c.). Sixty min
after oral treatment or thirty min after i.p. or s.c. injection,
the mice received a 20 µl intraplantar injection of acidiꢂed
saline (2% acetic acid in 0.9% saline, pH 2.0) into the plantar
surface of the right hind paw. After the injection of acidiꢂed
saline, the mice were placed into an acrylic box and the lick-
ing time was assessed for 20 min. The results were expressed
as the mean ± S.E.M of the licking time (s) (Meotti et al.
2
010; Rios et al. 2013). To verify the involvement of opioid
Antihyperalgesic activity The mice (n=10) were treated
receptors in the eꢁects of LQFM-096 on acid-induced noci-
ception, mice were pretreated with naloxone (3 mg/kg, i.p.)
or saline (10 ml/kg, i.p.) ꢂfteen min prior to treatment with
vehicle (10 ml/kg, 10% DMSO, p.o.), LQFM-096 (20 mg/
kg, p.o.), amiloride (100 mg/kg i.p.), or morphine (5 mg/
kg, s.c.).
with vehicle (10% DMSO, 10 ml/kg, p.o.), indomethacin
(
10 mg/kg, p.o.) or LQFM-096 (20 mg/kg, p.o.). Sixty min
after oral administration, carrageenan (50 µl) or prostaglan-
din E (PGE ) (50 μl, 100 ng/paw) was injected into the plan-
2
2
tar surface of the right hind paw or saline into the contralat-
eral paw. A progressively increasing pressure was applied
to the dorsal surface of the inꢀamed and uninꢀamed hind
paw until a withdrawal reꢀex was elicited. The nociceptive
threshold was evaluated from the diꢁerence between the
two paws at 1, 2, 3, and 4 h using an analgesimeter (Insight
Apparatus EFF-440—Brazil). A 400 g cut-oꢁ was set to
prevent paw lesions. A baseline (at time 0) was performed
for each animal before the treatments (Randall and Selitto
TRPV Channel Involvement The involvement of TRPV
channels in the antinociceptive eꢁects of LQFM-096 was
assessed through capsaicin-induced pain. Groups of mice
(n = 8) were treated with vehicle (10% DMSO, 10 ml/kg,
p.o.), LQFM-096 (20 mg/kg, p.o.) or morphine (5 mg/
kg s.c.). Sixty min after oral treatment or thirty min after
s.c. injection, the mice received a 20 µl intraplantar injec-
tion of capsaicin (1.6 µg/paw) into the plantar surface of the
right hind paw. After the capsaicin injection, the mice were
placed into an acrylic box and the licking time was assessed
for 7 min. The results were expressed as the mean± S.E.M
of the licking time (s) (Mohd Sani et al. 2012; Sakurada
et al. 1992). To verify the involvement of opioid receptors in
the eꢁect of LQFM-096 on capsaicin-induced pain, experi-
mental groups of mice were pretreated with naloxone (3 mg/
kg, i.p.) or saline (10 ml/kg, i.p.) ꢂfteen min prior to treat-
ment with vehicle (10 ml/kg, 10% DMSO, p.o.), LQFM-096
(20 mg/kg, p.o.) or morphine (5 mg/kg, s.c.).
1
957; Barbosa et al. 2013). The results were expressed as
the mean± S.E.M of the response to mechanical stimulus
in grams (g).
Study oꢁ the antinociceptive mechanism oꢁ action Opioid
receptor involvement The mice (n=8) were pretreated with
saline (10 ml/kg, i.p.) or naloxone (3 mg/kg, i.p.). After
ꢂfteen min, the animals were treated with vehicle (10%
DMSO, 10 ml/kg, p.o.), LQFM-096 (20 mg/kg, p.o.) or
morphine (5 mg/kg, s.c.). Sixty min after oral treatment or
thirty min after s.c. injection, the mice received formalin
K
ATP
Channel Involvement To investigate the role of
ATP
(
3%, v/v). The licking time was observed and recorded as
K
channels in the antinociceptive eꢁect of LQFM-096,
described in section “Formalin-induced nociception”. The
mice (n = 8) were pretreated with saline (10 ml/kg, i.p.)
1
3

Mechanisms involved in the antinociceptive and anti‑infammatory eꢀects oꢁ a new triazole…
or glibenclamide (3 mg/kg, i.p., selective KATP channel
blocker). After ꢂfteen min, the animals were treated with
vehicle (10 ml/kg, 10% DMSO, p.o.) or LQFM-096 (20 mg/
kg, p.o.). After sixty min, the mice received formalin (3%,
v/v) and the licking time was observed as described in sec-
tion “Formalin-induced nociception”. The results were
expressed as the mean± S.E.M of the licking time (s).
(w/v) sodium azide. The samples were subsequently centri-
fuged for 5 min at 300 g. The supernatant (100 μl) was trans-
ferred to a microplate well and the absorbance was moni-
tored at a wavelength of 450 nm. The results were expressed
as the mean± S.E.M of the enzymatic activity (mU/ml).
Measurement oꢁ TNF‑α levels After collection of the pleu-
ral exudate samples, the TNF-α levels were assessed by
enzyme-linked immunosorbent assay (ELISA) using a com-
Anti‑infammatory activity
®
mercial kit (Ebioscience ) according to manufacturer’s
Carrageenan‑induced paw oedema This test was per-
formed as described by Passos et al. (2007). Mice (n=10)
were treated with vehicle (10 ml/kg, 10% DMSO, p.o.),
indomethacin (10 mg/kg, p.o.) or LQFM-096 (20 mg/kg,
p.o.). After sixty min, the mice received an intraplantar
injection of 50 µl of carrageenan 1% in saline in the right
paw. The posterior left paw (control) received an injec-
tion of saline solution (0.9%) at the same volume. The paw
oedema was evaluated by the diꢁerences in the paw volume
using a plethysmometer at 1, 2, 3 and 4 h after carrageenan
injection. Baseline values were obtained before the treat-
ments. The results were expressed as the mean± S.E.M of
the diꢁerence in the paws in microliters.
instructions. Samples were collected 4 h after the induction
of pleurisy and centrifuged at 1200 g for 10 min at 4 °C. The
supernatant was later separated and stored at −70 °C. The
results were expressed as the mean± S.E.M of the TNF- α
levels in the pleural exudate (pg/ml).
Measurement oꢁ prostaglandin E (PGE ) The concentra-
2
2
tion of PGE in pleural exudate samples was determined by
2
ELISA using a commercial kit (Cayman Chemical). Sam-
ples were collected 4 h after the induction of pleurisy, with
PBS containing 10 μM indomethacin, and then were cen-
trifuged at 1200 g for 4 min at 4 °C, followed by separa-
tion and storage (−70 °C) of the supernatant until the assay.
Then, the basal PGE values were removed from all of the
2
Carrageenan‑induced pleurisy The pleurisy test was per-
samples (Florentino et al. 2017). Results were expressed as
formed according to the method described by Saleh et al.
the mean± S.E.M of the PGE levels in the pleural exudate
2
(
1999). The experimental groups (n=8) were treated with
(ng/ml).
vehicle (10 ml/kg, 10% DMSO, p.o.), dexamethasone
2 mg/kg, p.o.) or LQFM-096 (20 mg/kg, p.o.). Sixty min
(
CFA‑induced arthritis This experiment was performed
as described by Chopade and Sayyad (2013). Mice were
immunized with an intradermal injection of 50 µl of Com-
plete Freund’s Adjuvant (CFA) (0.5 mg/ml, Mycobacterium
butyricum) or 50 µl of saline approximately 1.5 cm distal
from the base of the tail. After 7 days, the animals (n=10)
were subjected to single oral and daily treatment with vehi-
cle (10 ml/kg, 10% DMSO), dexamethasone (2 mg/kg) or
LQFM-096 (20 mg/kg) for 14 days. One hour after the ꢂrst
treatment, a monoarthritic reaction was obtained through a
second injection of 50 µl of CFA (0.5 mg/ml, Mycobacte-
rium butyricum) or 50 µl of saline to the right hind paw.
The oedema and hyperalgesia were measured 1 h after CFA
injection into the paw. These measurements were repeated
every 2 days. After measuring the hyperalgesia and oedema
on day fourteen, the mice were euthanized by decapitation
and the paws were collected to quantify cellular migration
through histopathological analysis.
later, the animals were anaesthetized (2–3% halothane in
1
00% O ) prior to the injection of carrageenan (100 µl of
2
1
% in saline) into the pleural cavity (Moore 2003). Four
hours after carrageenan injection, the animals were eutha-
nized and the pleural exudate was collected with 1 ml of
heparinized phosphate-buꢁered saline. An aliquot was used
to count the number of total leukocytes using Türk solution
in a Neubauer chamber. Diꢁerential counting of leukocyte
mononuclear and polymorphonuclear cells was also carried
out. The results of the total and diꢁerential counting were
expressed as the mean± S.E.M of the absolute number of
total cells and each cell type, respectively. The ꢂnal result
was adjusted according to the unit of volume and expressed
6
as the number of leukocytes×10 /ml. An additional aliquot
of pleural exudate was used to determine the myeloperoxi-
dase (MPO) activity and quantify the tumour necrosis factor
alpha (TNF-α) levels.
Histopathological analysis The paws were fixed by
immersion in Metacarn (proportion: 60% methanol, 30%
chloroform, and 10% acetic acid) for 3 h. After ꢂxation,
the tissues were dehydrated in ethanol, diaphanized in
xylene, and embedded in paraplast (Histosec, Merck,
Darmstadt, Germany). The tissues were later sectioned
into 5 µm slices with a manual microtome (Leica RM2155,
Evaluation oꢁ MPO activity MPO activity was determined as
described previously (Saleh et al. 1999; Sedgwick 1995).
The pleural ꢀuid samples (20 μl) were added to 360 μl of
phosphate buꢁer containing 0.167 mg/ml of o-dianisidine
2
HCl and 0.0005% H O at pH 6.0. The enzyme reaction
2 2
was stopped after 15 min by the addition of 20 μl of 1%
1
3

C. S. Cardoso et al.
Nussloch, Germany). The sections were stained with haema-
toxylin–eosin (HE) for the quantiꢂcation of cell inꢂltration.
Thirty photomicrograph ꢂelds under a 40× objective were
obtained for analysis (6 ꢂelds/animal; n=5 animals/group).
The absolute number of mononuclear and polymorphonu-
clear cells per photomicrograph ꢂeld was obtained from
the photomicrographs. An A1 light microscope (Carl Zeiss
Microscopy, Gottingen, Germany) was used for the analysis
and the images were scanned using the ZEN2012-1.1.2.0
software (Zeiss) and Image-Pro Plus version 6.1 for Win-
dows (Media Cybernetics Inc., Silver Spring, MD, USA).
cycloaddition between 1-(4-ꢀuorophenyl)-1H-1,2,3-triazole-
4-carbonitrile (6) and NaN , using NH Cl as the catalyst
3
4
in DMF under microwave irradiation for 20 min with an
88% yield (Alterman and Hallberg 2000; Zwaagstra et al.
1997). When we used the same experimental conditions, but
with conventional heat, (2) was obtained after 72 h at 88%
yield (Zwaagstra et al. 1997). The synthetic route used a
green chemistry approach that had an atom economy greater
than 80% in each step; a green solvent, i.e., water; and a
microwave-assisted organic synthesis (MAOS) (Roschangar,
Sheldon, and Senanayake 2015; Trost 1991). LQFM-096
was obtained at 77% overall yield after three steps.
Statistical analysis
The data were statistically analysed by a one-way ANOVA
followed by Newman–Keul’s post hoc test or by two-way
ANOVA followed by Bonferroni’s post hoc test (Sokal and
Rohlf 1981). All statistical analyses were carried out using
GraphPad Prism version 5.00 and the data were expressed
as the mean ± S.E.M. Values of P ≤ 0.05 were considered
signiꢂcant.
Pharmacology
Antinociceptive activity oꢁ LQFM‑096
Acetic acid‑induced abdominal writhing As shown in
Fig. 3, oral treatment with LQFM-096 (10, 20, or 40 mg/
kg) decreased the number of writhes in a dose-dependent
manner [30.8% (P<0.001), 42.3% (P<0.001), and 51.3%
(
P<0.001), respectively] when compared to the con-
Results
trol group (95.1± 5.0). Indomethacin (positive control)
decreased the number of writhes by 40% (P<0.001).
Synthesis of 5‑(1‑(4‑ꢀuorophenyl)‑1H‑1,2,3‑triazol
‑
4‑yl)‑1H‑tetrazole—(LQFM‑096)
Formalin‑induced nociception In the formalin test, oral
treatment with LQFM-096 (20 mg/kg) reduced the formalin-
induced paw licking time in both the neurogenic and inꢀam-
matory phases compared to the control group (67.6± 4.7 s
As illustrated in Fig. 2, the synthetic route began with
-azido-4-ꢀuorobenzene (4) and proceeded through the
1
diazotization of 4-ꢀuoroaniline (3) in a quantitative yield,
which was used without puriꢂcation in the next step (L’abbé
et al. 1990). In turn, 1.3-bipolar cycloadditions were carried
out between 1-azido-4-ꢀuorobenzene (4) and 2-chloroacry-
lonitrile (5) in water to oꢁer 1-(4-ꢀuorophenyl)-1H-1,2,3-
triazole-4-carbonitrile (6) at 88% yield (Portman 1998).
st
nd
in the 1 phase and 221.4± 19.8 s in the 2 phase). LQFM-
096 reduced the licking time by 56.5% (P<0.01) and 43.1%
(P<0.01) in the ꢂrst and second phases, respectively. Indo-
methacin decreased the licking time in the second phase
(55%, P<0.001), while morphine decreased this parameter
in the ꢂrst (90.4%, P<0.001) and second (95.7%, P<0.001)
phases (Fig. 4).
5
-(1-(4-ꢀuorophenyl)-1H-1,2,3-triazol-4-yl)-1H-tetrazole—
(
LQFM-096) was synthesized through another 1.3-bipolar
Fig. 2 Synthetic route for the preparation of 5-(1-(4-ꢀuorophenyl)-1H-1,2,3-triazol-4-yl)-1H-tetrazole (2) (LQFM-096)
1
3

Mechanisms involved in the antinociceptive and anti‑infammatory eꢀects oꢁ a new triazole…
in the 4th hour). Indomethacin (positive control) also
reduced the nociceptive threshold between the paws. In the
1
50
00
PGE -induced hyperalgesia, LQFM-096 reduced the noci-
2
ceptive threshold between the paws by 86.6% (P<0.001)
in the 1st hour, 75.6% (P<0.001) in the 2nd hour, 80%
1
(
P<0.001) in the 3rd hour and 80.8% (P<0.001) in the
***
***
***
4
th hour compared to the control group (149.0± 18.6 g,
***
5
0
155.1± 6.7 g, 205.9± 5.7 g, and 206.9± 8.5 g in the 1st,
2
nd, 3rd and 4th hour of the test, respectively). Indometha-
cin did not inhibit the hyperalgesia (P>0.05; Fig. 5b).
0
1
0
20
40
Antinociceptive mechanism oꢁ LQFM‑096 Opioid recep-
tor involvement Administration of the nonselective opioid
receptor antagonist naloxone (3 mg/kg, i.p.) did not, per
se, aꢁect formalin-induced nociception. This compound
only antagonized the antinociceptive eꢁect of LQFM-
Vehicle
0 ml/kg
Indomethacin
10 mg/kg
LQFM-096 (mg/kg)
1
Fig. 3 Antinociceptive eꢁect of LQFM-096 (10, 20, 40 mg/kg, p.o.)
in the acetic acid-induced abdominal writhing test, in mice (n=8)
for 30 min. Indomethacin was used as a positive control. The verti-
cal bars represent the mean± S.E.M of the number of writhing for
0
96 (20 mg/kg p.o.) in the ꢂrst phase of the formalin test
(
P>0.05) and reversed the antinociceptive eꢁect of mor-
phine in both phases (Fig. 6). In the PGE -induced hyper-
3
0 min. ***P≤0.001 compared to the control group, according to
2
one-way ANOVA followed by the Newman Keuls’ post-test
algesia, naloxone (3 mg/kg, i.p.) attenuated the antihyper-
algesic eꢁect of LQFM-096 (20 mg/kg, p.o.) or morphine
(5 mg/kg, s.c.) (P>0.05) (Table 1).
Antihyperalgesic eꢀect oꢁ LQFM‑096 As shown in Fig. 5a,
ASIC Involvement Oral treatment with LQFM-096
oral treatment with LQFM-096 (20 mg/kg) or indomethacin
(20 mg/kg) elicited a signiꢂcant antinociceptive eꢁect with
a 53% reduction in the licking time compared to the control
group (229± 9.4 s; P<0.001; Fig. 7a). Amiloride (positive
control) decreased the licking time by 85.1% (P < 0.001;
Fig. 8a). Unlike amiloride, the antinociceptive eꢁect of
LQFM-096 (P > 0.05) was blocked by pretreatment with
naloxone (3 mg/kg, i.p.).
(
10 mg/kg) reduced the nociceptive threshold between the
uninꢀamed and inꢀamed paws in response to mechanical
stimuli throughout the test. LQFM-096 reduced the nocic-
eptive threshold between the paws by 23% (P<0.001), 28%
(
P<0.001), 30% (P<0.001), and 33% (P<0.001) in the
1
st, 2nd, 3rd and 4th hour of the test, respectively, compared
to the control group (152± 3.3 g in the 1st hour, 156± 2.7 g
in the 2nd hour, 154± 2.7 g in the 3rd hour, and 150± 3.0 g
TRPV Channel Involvement As shown in Fig. 7b,
capsaicin-induced pain was reduced in mice treated with
A
B
1
00
st
300
200
100
0
nd
1
Phase
2
Phase
5
0
**
**
***
***
0
Vehicle
0 ml/kg
LQFM-096
20 mg/kg
Indomethacin Morphine
0 mg/kg 5 mg/kg
Vehicle LQFM-096
Indomethacin Morphine
5 mg/kg
1
1
1
0 ml/kg
20 mg/kg
10 mg/kg
Fig. 4 Antinociceptive eꢁect of LQFM-096 (20 mg/kg, p.o.) in the
ꢂrst phase (a) and second phase (b) of formalin test, in mice (n=8).
Indomethacin and morphine were used as positive controls, during
the ꢂrst (0–5 min) and second phase (15–30 min). Vertical bars rep-
resent mean± S.E.M of licking time (s). **P≤0.01 and ***P≤0.001
compared to control group, according to one-way ANOVA followed
by Newman Keuls’ post- test
1
3

C. S. Cardoso et al.
A
A
1st Phase
*
**
1
80
50
20
1
00
###
#
##
*
**
1
8
0
1
***
*
**
*
**
*
**
6
0
9
0
*
**
6
0
***
40
***
***
3
0
2
0
0
0
0
1
2
3
4
Saline Naloxone Saline Naloxone Saline Naloxone
Vehícle
Time (Hours)
LQFM-096
20 mg/kg
Morphine
5 mg/kg
1
0 ml/kg
Vehicle 10 ml/kg, p.o.
LQFM-096 20 mg/kg, p.o.
Indomethacin 10 mg/kg, p.o.
B
2nd Phase
***
###
2
50
00
50
100
B
***
2
2
2
1
1
50
1
00
50
00
50
5
0
***
***
***
0
***
Saline Naloxone Saline Naloxone Saline
Naloxone
0
Vehicle
0 ml/kg
LQFM-096
20 mg/kg
Morphine
5 mg/kg
1
0
1
2
3
4
Time (Hours)
Vehicle 10 ml/kg, p.o.
Fig. 6 Eꢁect of pre-treatment with saline (10 ml/kg i.p.) or the non-
selective opioid receptor antagonist naloxone (3 mg/kg i.p.) on the
LQFM-096 (20 mg/kg p.o.) antinociceptive eꢁect in the ꢂrst phase
LQFM-096 20 mg/kg, p.o.
Indomethacin 10 mg/kg p.o.
(
a) and second phase (b) of the formalin test, in mice (n=8). Ver-
tical bars represent mean± S.E.M of licking time (s). ***P≤0.001,
#
##
compared to vehicle that received saline or P≤0.001 compared to
group treated that received saline, according to one-way ANOVA fol-
lowed by post hoc Newman Keuls’test
Fig. 5 Eꢁect of LQFM-096 (20 mg/kg, p.o.) on hyperalgesia induced
by carrageenan (a) and PGE (b), in mice (n=10). Indometha-
2
cin (10 mg/kg, p.o.) was used as positive control. The values were
expressed as mean± S.E.M of the diꢁerence of nociceptive threshold
between the non-inꢀamed and inꢀamed paw of animals in response
to mechanical stimulus (g). ***P≤0.001, compared to control group,
according to two-way ANOVA followed by Bonferroni’s post-test
+
glibenclamide (3 mg/kg, i.p.—ATP-sensitive K channel
inhibitor). Glibenclamide did not change the eꢁect of for-
malin-induced nociception per se. Unlike the second phase
of the formalin test, the antinociceptive eꢁect of LQFM-
0
96 (20 mg/kg p.o.) was only blocked by glibenclamide
LQFM-096 (20 mg/kg, p.o.). LQFM-096 induced a 33.8%
inhibition in the licking time compared to the control group
(P>0.05) in the ꢂrst phase, as is shown in Fig. 8.
(
59.4± 2.1 s; P<0.001). Pretreatment with naloxone (3 mg/
Anti‑infammatory activity
kg, i.p.) reversed the antinociceptive eꢁect of LQFM-096.
Channel Involvement The role of K channels was
K
Carrageenan‑induced paw oedema Oral treatment with
ATP
ATP
evaluated in the formalin test through the administration of
LQFM-096 (20 mg/kg) reduced the volume of paw oedema
1
3

Mechanisms involved in the antinociceptive and anti‑infammatory eꢀects oꢁ a new triazole…
Table 1 Involvement of opioid receptors in the anti-hyperalgesic
A
eꢁect of LQFM-096 (2) in PGE -induced hyperalgesia test, in mice
2
(
n=10)
300
***
**
Diꢁerence between the nociceptive thresholds of paws (g)
Mean± SEM)
*
***
(
Saline
Naloxone
200
1
2
3
4
st hour
Vehicle 10 ml/kg, p.o.
LQFM-096 20 mg/kg, p.o.
Morphine 5 mg/kg, s.c.
nd hour
Vehicle 10 ml/kg, p.o.
LQFM-096 20 mg/kg, p.o.
Morphine 5 mg/kg, s.c.
th hour
Vehicle 10 ml/kg, p.o.
LQFM-096 20 mg/kg, p.o.
Morphine 5 mg/kg, s.c.
th hour
Vehicle 10 ml/kg, p.o.
LQFM-096 20 mg/kg, p.o.
Morphine 5 mg/kg, s.c.
180.6± 12.8
14.7± 2.6***
5.7± 1.6***
136.5± 11.2
103.0± 10.2
151.2± 13.4
#
#
##
##
100
0
196.3± 11.7
13.4± 2.1***
9.4± 2.03*** 160.1± 14.8
189.4± 8.8
141.8± 12.6
#
#
##
##
Sal Nal Sal Nal Sal Nal Sal Nal
184.6± 11.8
12.7± 1.8***
13.8± 5.0***
185.2± 12.2
173.9± 15.8
189± 17.3
Morphine
Vehicle LQFM-096 Amiloride
#
##
##
10ml/kg 20 mg/kg 100 mg/kg 5 mg/kg
#
B
198.6± 12.0
17.2± 1.8***
28.4± 3.9***
206.0± 8.1
176.1± 13.4
183.3± 13.1
80
#
#
##
##
***
***
***
60
Values are expressed as mean± S.E.M. of the diꢁerence of nocicep-
tive threshold between the non-inꢀamed and inꢀamed paw of animals
in response to mechanical stimulus (g). ***P≤0.001, compared to
40
#
##
vehicle that received saline or P≤0.001 compared to group treated
that received saline, according to two-way ANOVA followed by Bon-
ferroni’s post-test
20
by 19.6% (P<0.001), 40.1% (P<0.001), 40.5% (P<0.001)
and 36.2% (P<0.001) in the 1st, 2nd, 3rd and 4th hour,
respectively, compared to the control group (118.9± 4.8 µl,
0
Sal
Vehicle
0 ml/kg
Nal
Sal
Nal
Sal
Nal
LQFM-096
20 mg/kg
Morphine
5 mg/kg
1
1
1
31.4± 8.3 µl, 123.3± 9.0 µl and 119.0± 5.0 µl in the
st, 2nd, 3rd and 4th hour, respectively). Indomethacin
Fig. 7 Eꢁect of pre-treatment with saline (10 ml/kg i.p.) or the non-
selective opioid receptor antagonist naloxone (3 mg/kg i.p.) on the
LQFM-096 (20 mg/kg p.o.) antinociceptive eꢁect in acidiꢂed saline
test (a) and capsaicin- induced nociception test (b), in mice (n=8).
Amiloride and morphine were used as positive controls. Vertical bars
represent mean± S.E.M of licking time (s). ***P≤0.001 compared
to control group or naloxone treated group, according to one-way
ANOVA followed by post hoc Newman–Keuls’ test
(
10 mg/kg, p.o.) reduced oedema by 39.2%, 49.3%, 47.3%
and 50.4% in the 1st, 2nd, 3rd and 4th hour, respectively
P<0.001; Fig. 9a).
(
Carrageenan‑induced pleurisy In the carrageenan-
induced pleurisy test, LQFM-096 (20 mg/kg, p.o.) reduced
the number of total leukocytes that migrated to the pleural
cavity by 41% (P < 0.001) compared to the control group
6
(
9.5 ± 0.3 × 10 leukocytes/ml). Dexamethasone (2 mg/kg,
Evaluation oꢁ MPO activity Oral treatment with LQFM-096
(20 mg/kg) inhibited MPO activity by 39.3% (P<0.01)
compared with the control group (74.8± 9.1 mU/ml). Simi-
larly, treatment with dexamethasone (2 mg/kg, p.o., positive
control) also reduced the activity of this enzyme by 76.7%
(P<0.001; Table 2).
p.o., positive control) reduced cell migration by 67.6%.
The diꢁerential count showed that the reduction in cel-
lular migration was due to a decrease in the number of
polymorphonuclear cells. The data in Table 2 showed
that LQFM-096 or dexamethasone reduced the number of
polymorphonuclear cells to 2.5 ± 0.2 and 0.6 ± 0.2 poly-
6
morphonuclear cells/ml × 10 , respectively, compared to
Measurement oꢁ TNF‑α levels Oral administration of
LQFM-096 (20 mg/kg) reduced the TNF-α levels by 24.4%
(P<0.05) compared with the control group (107.9± 8.7 pg/
the control group (6.5 ± 0.3 polymorphonuclear cells/
6
ml × 10 ).
1
3

C. S. Cardoso et al.
1st Phase
CFA‑induced arthritis The CFA-induced hyperalgesia in
this model was reduced by LQFM-096 (20 mg/kg, p.o.)
or dexamethasone (2 mg/kg, p.o.) in all the assessments
A
1
00
(
Fig. 9b). Although dexamethasone reduced the oedema
8
6
4
2
0
0
0
0
0
in all the evaluations, treatment with LQFM-096 did not
inhibit oedema formation (Fig. 9c).
#
##
Histopathological analysis The histopathological analysis
showed an increase in cell inꢂltration, mainly by polymor-
phonuclear cells, in the paw dermis of animals treated with
vehicle compared with the uninjured group. Administration
of LQFM-096 or dexamethasone reduced the inꢂltration of
polymorphonuclear cells (Figs. 9d, 10).
***
Saline
Glibenclamide Saline Glibenclamide
Vehicle
0 ml/kg
LQFM-096
20 mg/kg
1
Discussion
2nd Phase
B
The design, synthesis, and pharmacological evaluation of a
new triazole derivative, LQFM-096, were reported in this
study. This compound elicited antinociceptive eꢁects that
were mediated by the opioid/KATP and ASIC/TRPV1 path-
ways. Our ꢂndings also demonstrated the anti-inꢀammatory
eꢁects of this compound as well as reduced cell migration,
MPO activity and TNF-α levels. The doses of LQFM-096 in
this study were extrapolated from previous experiments with
LQFM-020 (LQFM-096 precursor) (Oliveira et al. 2016).
To investigate the antinociceptive eꢁects of LQFM-096,
the acetic-acid-induced abdominal writhing test was per-
formed. In general, acetic acid induces nociception through
the release of mediators (such as bradykinin, pro-inꢀamma-
tory cytokines, serotonin, histamine, and prostaglandins) and
the activation of ASICs, TRPV1, and glutamate receptors
2
50
2
1
1
00
50
00
*
**
50
0
Saline
Glibenclamide
Vehicle
Saline Glibenclamide
LQFM-096
20 mg/kg
1
0 ml/kg
(Costa et al. 2013; Gregory et al. 2013). The LQFM-096-in-
duced reduction in the number of writhes suggests antino-
ciceptive activity. Since LQFM-096 treatment at 40 mg/kg
Fig. 8 Eꢁect of pre-treatment with saline (10 ml/kg i.p.) or the selec-
ATP
+
tive K
antagonist glibenclamide (3 mg/kg i.p.) on the LQFM-096
(
highest dose) did not induce a signiꢂcant change in the bar-
(
20 mg/kg p.o.) antinociceptive eꢁect in the ꢂrst phase (a) and second
phase (b) of the formalin test, in mice (n=8). Vertical bars represent
mean± S.E.M of pain reaction time (s). ***P≤0.001, compared to
biturate sleep and open ꢂeld tests, the antinociceptive eꢁects
of this compound are unlikely to compromise motor activity
or be associated with sedation (Supplementary Material). In
subsequent evaluations, the intermediate dose of LQFM-096
with a margin of safety was adopted to reduce the number of
experimental animals.
#
##
vehicle that received saline or P≤0.001 compared to group treated
that received saline, according to one-way ANOVA followed by New-
man Keuls’ post-test
ml). Dexamethasone (2 mg/kg, p.o., positive control) also
The formalin test was also employed to conꢂrm the antin-
ociceptive eꢁects of LQFM-096. Neurogenic and inꢀam-
matory pain can be evaluated in the ꢂrst and second phases
of this test, respectively (Hanskaar and Hole 1987; Shibata
et al. 1989). The reduction in the licking time in both phases
of this test conꢂrmed the antinociceptive eꢁects of LQFM-
096. The result obtained with LQFM-096 against neuro-
genic pain shows largar eꢁectiveness when compared with
the result obtained with LQFM-020 (Oliveira et al. 2016)
in the same experimental model, guiding this research to
reduced the TNF-α levels by 88.7% (P<0.001; Table 2).
Measurement oꢁ PGE levels PGE , a major product of
2
2
the enzymatic reaction catalyzed by COX-2 in inꢀamma-
tory process, was also analyzed. Compared with the con-
trol group (21.8± 0.2 ng/ml), treatment with LQFM-096
2
0 mg/kg showed signiꢂcant reduction of the PGE levels
2
by 16.5%. Dexamethasone showed the expected proꢂle and
reduced the PGE levels by 22% (Table 2).
2
1
3

Mechanisms involved in the antinociceptive and anti‑infammatory eꢀects oꢁ a new triazole…
A
B
1
50
250
00
50
00
2
*
100
1
*
**
***
***
***
***
**
*
**
*
***
1
***
*
**
*** ***
*
**
***
5
0
***
*
** *** ***
*
* *** ***
*
*
***
50
0
0
0
1
2
3
4
B 0
2
4 5 6
Time (Days)
8
10 12 14
Time (Hours)
Saline + Vehicle 10 ml/kg
CFA + Vehicle 10 ml/kg
Vehicle 10 ml/kg p.o.
LQFM-096 20 mg/kg p.o.
Indomethacin 10 mg/kg p.o.
CFA + LQFM-096 20 mg/kg
CFA + Dexamethasone 2 mg/kg
C
D
4
3
2
1
0
0
0
0
80
*
**
#
***
##
60
###
***
40
***
***
#
##
*
**
***
***
*
** *** *** ***
***
*
**
20
0
0
B 0
2
4
6
8
10 12 14
Time (Days)
Poly
Mono
Saline + Vehicle 10 ml/kg
CFA + Vehicle 10 ml/kg
Saline + Vehicle 10 ml/kg
CFA + Vehicle 10 ml/kg
CFA + LQFM-096 20 mg/kg
CFA + LQFM-096 20 mg/kg
CFA + Dexamethasone 2 mg/kg
CFA + Dexamethasone 2 mg/kg
Fig. 9 Eꢁect of LQFM-096 (20 mg/kg, p.o.) on acute and chronic
non-inꢀamed and inꢀamed paw of animals in response to mechanical
stimulus (g). (n=10). The vertical bars represent the mean± S.E.M
of mononuclear and polymorphonuclear leukocytes number that
migrates to the paw dermis from thirty photomicrographs ꢂelds per
group. ***P≤0.001, compared to control group not injured and
inꢀammation, in mice. The graphs show the eꢁect of LQFM-
0
96 (20 mg/kg, p.o.) on a carrageenan-induced paw oedema test
(
acute inꢀammation), b CFA-induced hyperalgesia, c CFA-induced
oedema and d cell quantiꢂcation obtained from histopathological
analysis of paw of animals submitted to the CFA-induced arthritis
test. Indomethacin (10 mg/kg, p.o.) and dexamethasone (2 mg/kg,
p.o.) were used as positive control. The graphics in line represent
the mean± S.E.M of the diꢁerence in volume between the paws, in
microliters or the diꢁerence of nociceptive threshold between the
#
##
P≤0.001, compared to control group injured, according to one-
way ANOVA followed by Newman–Keuls’ post-test or according to
two-way ANOVA followed by Bonferroni’s post-test. Mono mononu-
clear, Poly polymorphonuclear
elucidating the possible mechanisms of action involved in
this antinociceptive eꢁect.
of the primary nociceptive ꢂbres by inꢀammatory mediators
(Anseloni et al. 2003; Chandy and Moore 1998).
The elucidation of the mechanisms underlying the antino-
ciceptive activities of LQFM-096 was proposed to further
the pharmacological characterization of this new compound.
To verify whether LQFM-096-induced antinociception is
associated with anti-inꢀammatory activity, carrageenan- or
prostaglandin-induced hyperalgesia were carried out. Inꢀam-
mation often induces hyperalgesia through the sensitization
According to Tsuchida et al. (2015), carrageenan injec-
tion into an animal’s paw increased prostaglandin levels,
which in turn sensitize the nociceptive ꢂbres. PGE injec-
2
tion activates the EP1 and EP4 receptors in the nocicep-
tive ꢂbres. These receptors often induce intracellular signal
cascades that culminate in hyperalgesia (Kawabata 2001).
The experimental data showed that LQFM-096 inhibited
1
3

C. S. Cardoso et al.
Table 2 Anti-inꢀammatory
activity of LQFM-096 in
carrageenan-induced pleurisy
test, in mice (n=8)
Inꢀammatory Parameters
Vehicle
LQFM-096
Dexamethasone
2 mg/kg, p.o.
1
0 ml/kg, p.o.
20 mg/kg, p.o.
6
Total leukocytes×10 /ml
9.5± 0.3
6.5± 0.3
3.0± 0.3
74.8± 9.1
107.9± 8.7
21.8± 0.2
5.6± 0.6 ***
2.5± 0.2***
3.1± 0.2
45.4± 7.6**
81.6± 7.0*
18.2± 1.1***
3.1± 0.2***
0.6± 0.2***
2.9± 0.4
17.4± 2.6***
12.29± 3.1***
16.9± 0.7***
6
Polymorphonuclears cells×10 /ml
6
Mononuclears cells×10 /ml
MPO activity (mU/ml)
TNF-α levels (pg/ml)
PGE levels (ng/ml)
2
6
Values are expressed as mean± S.E.M. of total number of leukocytes×10 /ml, number of polymorpho-
6
6
nuclears cells×10 /ml and number of mononuclears cells×10 /ml migrated into the pleural cavity, as
well as the MPO activity (mU/ml), TNF-α levels (pg/ml) and PGE levels (ng/ml) in the pleural exudate.
2
*
P≤0.05, **P≤0.01 and ***P≤0.001, according one-way ANOVA followed by Student–Newman–
Keuls’ test
Fig. 10 Histopathology of the paw dermis of the control group
not injured (a), control group injured (b) and groups treated with
LQFM-096 (c) and dexamethasone (d). The absence of cellular inꢂl-
tration was observed in the group Saline+Vehicle (a). The group
CFA+Vehicle showed prominent inꢀammatory cells inꢂltration (b),
while LQFM-096 and dexamethasone were reduced this inꢂltration
(c, d). The photomicrographs were taken with 40×objective (Stain-
ing: hematoxylin–eosin). Scale bar of histology: 20 µm
1
3

Mechanisms involved in the antinociceptive and anti‑infammatory eꢀects oꢁ a new triazole…
carrageenan- or PGE -induced hyperalgesia. Hence, the
eꢁects of LQFM-096 suggest that inhibition of these chan-
2
antinociceptive eꢁects of LQFM-096 suggest the involve-
nels is involved in the activation of the opioid pathway.
Despite the potential roles of ion channels and the opi-
oid pathway in the antinociceptive activity of LQFM-096,
non-blockade of the second phase of the formalin test by
antagonists suggest that the analgesic eꢁects of this com-
pound involve an anti-inꢀammatory component. According
to Giorno et al. (2016), the antinociceptive eꢁect in the ꢂrst
phase is associated with a direct action on the opioid path-
way while the antinociceptive eꢁect in the second phase
involves the inhibition of inꢀammatory mediators.
ment of mechanisms that are independent of inhibitory
actions on PGE synthesis.
2
Opioid receptors play important roles in analgesia by
inhibiting adenylate cyclase, reducing cAMP, and releas-
ing neurotransmitters, among others (Sehgal et al. 2011). In
this manner, we assessed the participation of opioid recep-
tors in the antinociceptive eꢁects of LQFM-096. In PGE2-
induced hyperalgesia, the eꢁect of LQFM-096 was blocked
by naloxone. Additional characterization of this eꢁect was
performed in the formalin test with naloxone pretreatment.
The results showed that naloxone only blocked the antinocic-
eptive eꢁects of LQFM-096 in the ꢂrst phase of the formalin
test. Thus, the antinociceptive eꢁects appear to be partially
mediated by opioid receptors. Opioid receptor activation
The anti-inꢀammatory actions of LQFM-096 were inves-
tigated through carrageenan-induced paw oedema and pleu-
risy. Carrageenan induces inꢀammation by releasing media-
tors such as histamine, serotonin, bradykinin, prostaglandins
and nitric oxide (Di Rosa 1972; Passos et al. 2007). In these
tests, LQFM-096 elicited anti-inꢀammatory activities by
reducing the recruitment of polymorphonuclear cells (such
as neutrophils) and suppressing oedema formation.
+
+
promotes the opening of K channels, K eꢃux and cellu-
lar hyperpolarization (Campbell and Welch 2001). Thus, we
+
investigated the participation of the ATP-sensitive K chan-
+
nel by using glibenclamide (ATP-sensitive K channel inhib-
Neutrophil granules are rich in MPO. This enzyme often
interacts with hydrogen peroxide and chloride to generate
hypochlorous acid inside the phagosome, thus eliminating
the aggressor agent (Klebanoꢁ 2005). LQFM-096 reduced
the MPO activity. This result corroborates the reduction in
polymorphonuclear cells in the previous study (Haegens
et al. 2009).
itor). The antinociceptive eꢁect of LQFM-096 in the ꢂrst
phase of the formalin test was attenuated by pretreatment
with glibenclamide. These results conꢂrm the involvement
+
of the opioid/K
pathway in the antinociceptive eꢁect of
ATP
LQFM-096, which is consistent with that of PILAB 8 (tria-
zole compound) as reported by Montes et al. (2017). Thus,
1
,3,5-trisubstituted 1,2,4-triazoles have shown potent and
The involvement of TNF-α in the anti-inflammatory
actions of LQFM-096 was also assessed. TNF-α is a potent
inꢀammatory mediator that regulates the induction of adhe-
sion molecules in the endothelium and contribute to the
recruitment of leukocytes to inꢀammatory sites (Nourshargh
and Alon 2014; Zelová and Hosek 2013). In this study,
LQFM-096 reduced TNF-α levels. This eꢁect appears to
contribute to the reduction in cell migration, MPO activity,
and subsequent anti-inꢀammatory activity.
selective δ opioid receptor binding (Peng et al. 2009).
The ASICs and TRPV1 ion channels are involved in the
genesis of pain, particularly in inꢀammatory conditions with
tissue acidosis (Baggio et al. 2012; Deval et al. 2010; Ma and
Quirion 2007; Mickle et al. 2016). Hence, these channels
are important pharmacological targets for the development
of new analgesic drugs. Our ꢂndings showed that LQFM-
0
96 attenuated the nociception induced by acidiꢂed saline
(
an agonist of ASICs) or capsaicin (an agonist of TRPV1).
Furthermore, in this study, it was observed that LQFM-
These results suggest that LQFM-096 can modulate ASICs
and TRPV1 ion channels.
096 reduced the PGE levels in the pleural exudate, cor-
2
roborating with result obtained in the PGE -induced hyper-
2
A study carried out by Cai et al. (2014) showed that mor-
phine induced cAMP/PKA-dependent inhibition of ASIC
channel currents through opioid receptors. Additionally,
Endres-Becker et al. (2007) reported the µ-opioid-mediated
inhibition of the TRPV1 channel via Gi/o proteins and the
cAMP pathway. In this manner, we sought to verify whether
the interaction between LQFM-096 and these channels is
through activation of the opioid pathway. In the acidic
saline- and capsaicin-induced nociception, naloxone blocked
the antinociceptive eꢁects of LQFM-096. This result sug-
gests that the inhibition of these channels by LQFM-096 is
dependent on the opioid pathway.
algesia. Therefore, part of the anti-inꢀammatory eꢁect of
LQFM-096 involves the inhibition of PGE production.
2
In the presence of pro-inflammatory mediators, the
expression of ASICs and TRPV1 channels is increased.
This increase, in turn, could facilitate the development and
maintenance of inꢀammatory pain (Ma and Quirion 2007;
White et al. 2010). Therefore, the anti-inꢀammatory activity
of this compound may decrease the expression and activa-
tion of these channels. Hypothetically, the antinociceptive
eꢁects of LQFM-096 could be considered to be independent
of the opioid pathway if the latter assumption is true. This
hypothesis seems to be supported by the non-blockade eꢁect
of LQFM-096 in the second phase of the formalin test.
Finally, we investigated the eꢁects of LQFM-096 on
CFA-induced arthritis, which is a chronic inꢀammatory
The development of mechanical hyperalgesia has been
associated with the TRPV1 ion channels and ASICs (Wata-
nabe et al. 2015; White et al. 2010). The antihyperalgesic
1
3

C. S. Cardoso et al.
model. This is a well-established model for the evaluation of
substances with anti-arthritic potential, as it mimics arthritis
in clinical setup (Vijayalaxmi et al. 2015). This model is
associated with oedema formation and a reduction in the
nociceptive threshold (Rajkapoor et al. 2009). Although
treatment with LQFM-096 did not reduce the formation
of oedema, attenuation of hyperalgesia and a reduction in
polymorphonuclear cell inꢂltration in the paw dermis were
reported. These results showed that the anti-inꢀammatory
eꢁects of LQFM-096 were maintained in the chronic model.
extract, fractions and 8-methoxylapachenol from Sinningia alla-
gophylla tubers. Basic Clin Pharmacol Toxicol 113:1–7
Benyamin R, Trescot AM, Datta S, Buenaventura R, Adlaka R et al
(
2008) Opioid complications and side eꢁects. Pain Physician
11:S105–S120
Cai Q, Qiu C, Qiu F, Liu T, Qu Z et al (2014) Morphine inhibits
acid-sensing ion channel currents in rat dorsal root ganglion
neurons. Brain Res 1554:12–20. https://doi.org/10.1016/j.brain
res.2014.01.042
Campbell VC, Welch SP (2001) The role of minoxidil on endogenous
opioid peptides in the spinal cord: a putative co-agonist rela-
tionship between K-ATP openers and opioids. Eur J Pharmacol
4
17(1–2):91–98
Chandy RL, Moore PK (1998) Eꢁects of selective inhibitors of neu-
ronal nitric oxide synthase on carrageenan-induced mechanical
and thermal hyperalgesia. Neuropharmacology 37(1):37–43
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Conclusions
This study showed that oral administration of LQFM-096
exerts acute antinociceptive eꢁects. The attenuation of the
analgesic eꢁects of this compound by pretreatment with
naloxone and glibenclamide suggest that the antinocicep-
tive activity of this triazole derivative is likely mediated by
Costa EA, Lino RC, Gomes MN, Nascimento MNM, Florentino IF et al
(
2013) Anti-inꢀammatory and antinociceptive activities of LQFM
002—a 4-nerolidylcatechol derivate. Life Sci 92:237–244. https
://doi.org/10.1016/j.lfs.2012.12.003
Deval E, Lingueglia E (2015) Acid-sensing ion channels and nocic-
eption in the peripheral and central nervous systems. Neurop-
harmacology 94:49–57. https://doi.org/10.1016/j.neuropharm
.2015.02.009
the activation of opioid receptors and K
channels as well
ATP
as opioid-dependent inhibition of ASICs and TRPV1 ion
channels. The LQFM-096-induced anti-inꢀammatory eꢁect
Deval E, Gasull X, Noël J, Salinas M, Baron A et al (2010) Acid-
sensing ion channels (ASICs): pharmacology and implication in
pain. Pharmacol Ther 128:549–558. https://doi.org/10.1016/j.
pharmthera.2010.08.006
involves reduction in TNF-α and PGE levels, cell migra-
2
tion and MPO activity in animal models of inꢀammation.
Altogether, these ꢂndings suggest that LQFM-096 might be
a promising prototype for developing a new analgesic and
anti-inꢀammatory agents.
Dheer D, Singh V, Shankar R (2017) Medicinal attributes of 1,2,3-tria-
zoles: current developments. Bioorg Chem 71:30–54
Di Rosa M (1972) Biological properties of carrageenan. J Pharmacol
2
4(2):89–102
Endres-Becker J, Heppenstall PA, Mousa SA, Labuz D, Oksche A et al
Acknowledgments The authors are grateful to Dr. Ekaterina A. F.
B. Rivera, Lucas B. do Nascimento and Taís Andrade Dias de Souza
for ethical and technical assistance, as well as the CNPq (Conselho
Nacional de Desenvolvimento Cientíꢂco e Tecnológico), and CAPES
(
2007) Mu-opioid receptor activation modulates transient recep-
tor potential vanilloid 1 (TRPV1) currents in sensory neurons in
a model of inꢀammatory pain. Mol Pharmacol 71:12–18. https://
doi.org/10.1124/mol.106.026740
(
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for
Fein A (2011) Nociceptores: as células que sentem dor. Tradução de
PETROV, P.; FRANCISCHI, J. N.; FERREIRA, S. H. Ribeirão
Preto/SP. 2011. Disponível em: http://cell.uchc.edu/pdf/fein/nocic
eptors_fein_2012.pdf
ꢂnancial support.
Compliance with ethical standards
Florentino IF, Silva DPB, Silva DM, Cardoso CS, Moreira ALE,
Borges CL, Soares CMA, Galdino PM, Lião LM, Ghedini PC,
Menegatti R, Costa EA (2017) Potential anti-inꢀammatory eꢁect
of LQFM-021 in carrageenan-induced inꢀammation: the role of
nitric oxide. Nitric Oxide 69:35–44. https://doi.org/10.1016/j.
niox.2017.04.006
Conꢁlict oꢁ interest The authors declare no conꢀicts of interest.
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