New pyrazole derivative 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole: synthesis and assessment of some biological activities

Article abstract of DOI:10.1111/cbdd.12838

The molecular modification and synthesis of compounds is vital to discovering drugs with desirable pharmacological and toxicity profiles. In response to pyrazole compounds' antipyretic, analgesic, and anti-inflammatory effects, this study sought to evaluate the analgesic, anti-inflammatory, and vasorelaxant effects, as well as the mechanisms of action, of a new pyrazole derivative, 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole. During the acetic acid-induced abdominal writhing test, treatments with 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole reduced abdominal writhing, while during the formalin test, 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole reduced licking times in response to both neurogenic pain and inflammatory pain, all without demonstrating any antinociceptive effects, as revealed during the tail flick test. 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole also reduced carrageenan-induced paw edema and cell migration during the carrageenan-induced pleurisy test. As demonstrated by the model of the isolated organ, 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole exhibits a vasorelaxant effect attenuated by Nω-nitro-l-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, tetraethylammonium or glibenclamide. 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole also blocked CaCl₂-induced contraction in a dose-dependent manner. Suggesting a safe toxicity profile, 5-[1-(4-fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole reduced the viability of 3T3 cells at higher concentrations and was orally tolerated, despite signs of toxicity in doses of 2000?mg/kg. Lastly, the compounds' analgesic activity might be attributed to the involvement of the NO/cGMP pathway and K⁺ channels observed in the vasorelaxant effect.

Full text of DOI:10.1111/cbdd.12838

Received Date : 04-Apr-2016
Revised Date : 28-Jun-2016
Accepted Date : 06-Aug-2016
Article type : Research Article
New pyrazole derivative 5-[1-(4-fluorphenyl) -1H-pyrazol-4-yl]-2H-tetrazole: Synthesis and
assessment of some biological activities
Lanussy Porfiro de Oliveira,a Daiany Priscilla Bueno da Silva,a Iziara Ferreira Florentino,a James
Oluwagbamigbe Fajemiroye,a Thiago Sardinha de Oliveira,b Renato Ivan de Ávila Marcelino,c Francine
Pazini,d Luciano Morais Lião,e Paulo César Ghedini,b Soraia Santana de Moura,c Marize Campos
Valadares,c Verônica Vale de Carvalho,e Boniek Gontijo Vaz,e Ricardo Menegatti,d Elson Alves Costaa
a Laboratory of Pharmacology of Natural and Synthetic Products, Department of Pharmacology,
Institute of Biological Sciences, Federal University of Goiás, Campus Samambaia, Goiânia, GO, Brazil
bLaboratory of Biochemistry and Molecular Pharmacology, Department of Pharmacology, Institute of
Biological Sciences, Federal University of Goiás, Goiânia, GO, Brazil
cLaboratory of Cellular Pharmacology and Toxicology, FarmaTec, College of Pharmacy,
Federal University of Goiás, Goiânia, GO, Brazil
dLaboratory of Medicinal Pharmaceutical Chemistry, College of Pharmacy, Federal University of
Goiás, Goiânia, GO, Brazil
eChemistry Institute, Federal University of Goiás, Campus Samambaia, Goiânia, GO, Brazil
Corresponding author mail id:- xico@ufg.br
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12838
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Highlights
Given pyrazole compounds’ antipyretic, analgesic, and anti-inflammatory effects, we
describe the synthesis and pharmacotoxicological evaluation of a new pyrazole derivative, 5-[1-(4-
fluorphenyl)-1H-pyrazol-4-yl]-2H-tetrazole, as well as the antinociceptive, anti-inflammatory activity
and involvement of the NO/cGMP pathway and ionic channels in its vasorelaxant effect. The
blockade of voltage-dependent calcium channels could have also contributed to the vasorelaxant
effect of the compound, which demonstrated a low toxicological profile promising antinociceptive,
anti-inflammatory, and vasorelaxant effects.
Abstract
The molecular modification and synthesis of compounds is vital to discovering drugs with desirable
pharmacological and toxicity profiles. In response to pyrazole compounds’ antipyretic, analgesic, and
anti-inflammatory effects, this study sought to evaluate the analgesic, anti-inflammatory, and
vasorelaxant effects, as well as the mechanisms of action, of a new pyrazole derivative, 5-[1-(4-
fluorphenyl)-1H-pyrazol-4-yl]-2H-tetrazole (FPPT). During the acetic acid-induced abdominal writhing
test, treatments with FPPT reduced abdominal writhing, while during the formalin test, FPPT
reduced licking times in response to both neurogenic pain and inflammatory pain, all without
demonstrating any antinociceptive effects, as revealed during the tail flick test. FPPT also reduced
carrageenan-induced paw edema and cell migration during the carrageenan-induced pleurisy test.
As demonstrated by the model of the isolated organ, FPPT exhibits a vasorelaxant effect attenuated
by Nω-nitro-L-arginine methyl ester, 1H-[1,2, 4]oxadiazolo[4,3-alpha]quinoxalin-1-one,
tetraethylammonium or glibenclamide. FPPT also blocked CaCl2-induced contraction in a dose-
dependent manner. Suggesting a safe toxicity profile, FPPT reduced the viability of 3T3 cells at
higher concentrations and was orally tolerated, despite signs of toxicity in doses of 2,000 mg/kg.
Lastly, the compound’s analgesic activity might be attributed to the involvement of the NO/cGMP
pathway and K+ channels observed in the vasorelaxant effect.
Keywords: Pyrazole derivatives, antinociceptive effect, anti-inflammatory effect, vasorelaxant effect,
NO/cGMP pathway
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Introduction
A natural physiological response to the removal of harmful agents and repair of lesioned
tissues (1,2), inflammation is often accompanied by pain, typically as an adaptive alert mechanism
that triggers appropriate protective responses to real or imminent injury (3). However, at a certain
point, both pain and inflammation no longer serve the purpose of alerting and protecting the
organism, yet continue to significantly affect the lifestyles of millions of people worldwide (4–6).
Worse still, treating those symptoms with analgesics and anti-inflammatory drugs can cause side
effects, primarily in the gastrointestinal tract. In response, developing drugs with strong therapeutic
effects and weak side effects (7,8) remains crucial.
The pyrazole compound is a synthetic heterocycle with a five-membered ring, which
includes two adjacent nitrogen atoms, three carbon atoms, and two double bonds (9,10). Since
compounds with that chemical structure have demonstrated antimicrobial (11), antidepressant,
anticonvulsant (12), antipyretic (13), analgesic, and anti-inflammatory effects (14,15), the molecular
modification and synthesis of pyrazole derivatives could yield drug prototypes with desirable
pharmacological aspects and minimal toxicity. To that end, the pyrazole derivative 5-[1-(3-
fluorophenyl)-1H-pyrazol-4-yl]-2H-tetrazole (FPPT) was designed and synthesized from the
commercially available pyrazole derivatives milrinone (1) and cilostazol (2).
Martins et al. (2013) (16) recently demonstrated the endothelium-independent relaxation of
vascular smooth muscle 5-(1-(3-fluorophenyl)-1H-pyrazol-4)-2H-tetrazola (LQFM021), an analog of
FPPT, which involved the activity of K+ and Ca2+ channels, AC/cAMP, and GC/cGMP. In support, other
studies have shown that the L-arginine/NO/cGMP pathway and K+ and Ca2+ channels are involved in
the mechanisms of antinociceptive activity in drugs such as morphine, dipyrone, and diclofenac (17–
20). Opioids, for example, can inhibit neuronal activity by inhibiting voltage-dependent Ca2+ channels
in neurons of the dorsal root ganglion and by activating K+ channel rectifiers in postsynaptic neurons
in the spinal cord, thereby promoting an antinociceptive effect (20, 21).
Considering the analgesic and anti-inflammatory activity of pyrazole compounds associated
with ion channels, the aim of our study was to evaluate the antinociceptive, anti-inflammatory, and
vasorelaxant effects of the new pyrazole derivative FPPT and, more particularly, investigate the
involvement of the NO/cGMP/ pathway and both K+ and Ca2+ channels in its vasorelaxant effect. We
describe the synthesis and pharmacotoxicological evaluation of the new FPPT (4) heterocyclic
derivative, originally designed as regioisomers from the LQFM021 (3) compound described by
Martins et al. (2013) (16) and Florentino et al. (2015) (15), as illustrated in Figure 1A.
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Material and Methods
Analytical instruments
All melting points were determined with a Marte 284594 apparatus. Nuclear magnetic
resonance (NMR) data were recorded using a spectrometer (AVANCE III, Bruker, Atibaia-SP, Brazil)
operating at 500.13 MHz proton frequency. Samples for NMR measurements were prepared in CDCl3
containing 1% tetramethylsilane (TMS) as an internal standard. Splitting patterns were d (doublet)
and ddd (double double doublet). Infrared (IR) spectra were obtained with a Fourier transform (FT)-
IR instrument (Spectrum 400N, Perkin–Elmer, Waltham, USA) in KBr plates. Microanalytical data
were obtained with a spectrometric system (Spectrum BXII FT-IR, Perkin–Elmer) using a digital
analytical scale (Gehaka, São Paulo, Brazil), whereas mass spectra were obtained with a mass
spectrometer (Q-Exactive, Thermo Fisher Scientific, Bremen, Germany). The sample preparation for
mass spectrometric analysis consisted of diluting 1 mg of each sample in 1 mL of methanol with 0.1%
formic acid. The solution obtained was directly infused at a flow rate of 2 µL/min into the
electrospray ionization source, whose conditions were a nebulizer gas pressure of 0.5–1.0 bar, a
capillary voltage of 3.0 kV, and capillary transfer temperature of 250 °C. The progress of all reactions
was monitored by thin-layer chromatography performed on 2.0 × 6.0-cm aluminium sheets
precoated with silica gel 60 (Merck, Barueri-SP, Brazil) to a thickness of 0.25 mm. The developed
chromatograms were viewed under ultraviolet light (254–265 nm) and treated with iodine vapor.
For column chromatography, we used silica gel 70–230 mesh (Merck). Reagents and solvents were
purchased from commercial suppliers.
Animals
Experiments were performed using female and male Swiss albino mice (25–30 g) and male
Wistar rats (200–250 g) from the Central Animal House of the Federal University of Goiás, Goiânia,
Brazil. Animals were kept in plastic cages at 22 ± 2 °C under a 12-h light–dark cycle with free access
to pellet food and water in compliance with the International Guiding Principles for Biomedical
Research Involving Animals. Animals were acclimatized for 7 d before experiments commenced. All
experimental protocols were developed according to the principles of ethics and animal welfare
designated by the Ethics Committee on Animal Experimentation. Experimental protocols were
approved by the ethics committee of Federal University of Goiás (no. 137/2009, 017 and 020/2013).
After each experiment, animals were anesthetized with the mixture of xylazine (10 mg/kg) and
ketamine hydrochloride (100 mg/kg) administered intraperitoneally, prior to being euthanized by
cervical dislocation (22).
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Chemicals
Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, trypsin,
ethylenediaminetetraacetic acid (EDTA), neutral red, carrageenan, dimethyl sulfoxide (DMSO),
phenylephrine (PhE), acetylcholine (ACh), sodium nitroprusside, Nω-nitro-L-arginine methyl ester (L-
NAME), tetraethylammonium (TEA), and 1H-[1,2, 4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ)
were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetic acid, ethanol, and glacial acetic acid
were acquired from Vetec (Rio de Janeiro, RJ, Brazil), whereas naloxone chloridrate (Narcan®) and
morphine (Dimorf®) were obtained from Cristalia (São Paulo, SP, Brazil). Dexamethasone and
indomethacin were purchased from Prodome (Campinas, SP, Brazil), xylazine from Syntec (Cotia, SP,
Brazil), and ketamine from König (Santana de Parnaíba, SP, Brazil). Acetone, formaldehyde, heparin,
NaCl, and Türk solution were obtained from Isofar (Duque de Caxias, RJ, Brazil), Synth (Diadema, SP,
Brazil), Hipolabor (Belo Horizonte, MG, Brazil), New Prov (Pinhais, PR, Brazil), respectively.
Syntheses
Synthesis of 1-(3-Fluorophenyl)-1H-pyrazole-4-carbonitrile (9)
To a heterogeneous mixture of NH2OH·HCl (1.3 mmol) and NaI (4 mmol) in
dimethylformamide (DMF) (4 mL), 1-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde (8) (1 mmol) was
added and stirred at room temperature. The reaction mixture was heated at reflux temperature for
6 h and then cooled by pouring ice. The precipitate was vacuum filtered and dried, and the crude
product was purified by chromatography using CHCl3 as a mobile phase to provide 1-(4-
fluorophenyl)-1H-pyrazole-4-carbonitrile (9) as a beige solid in an 84% yield, as Figure 2A shows,
characterized as mp 162 °C, Rf = 0.65 (hexane:ethyl acetate, 7:3): IRmax (KBr) cm−1: 3,132 (ν C–H),
1
2,233 (ν C–N), and 838 (ν 1,3-C–F); H NMR (500.13 MHz) CDCl3 δ: 8.26 (1H, d, J = 0.6 Hz, H-5), 7.98
(1H, d, J = 0.6 Hz, H-3), 7.65 (1H, ddd, J = 9.3, 4.6, and 3.4 Hz, H-2′), 7.65 (1H, ddd, J = 10.3, 4.6, and
3.4 Hz, H-6′), 7.20 (1H, ddd, J = 9.3, 8.3, and 3.4 Hz, H-3′), 7.20 (1H, ddd, J = 10.3, 9.3, and 3.4 Hz, H-
5′); 13C NMR (125.76 MHz) CDCl3 δ: 162.0 (C-4′),143.3 (C-3), 135.0 (C-1′), 132.0 (C-5), 121.9 (C-2’ and
6′), 116.7 (C-3’ and 5′),112.8 (C–CN), 94.6 (C-4), and [M+H]+ m/z of 188.06166 (error = 1.0 ppm) .
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Synthesis of 5-(1-(4-Fluorophenyl)-1H-pyrazol-4-yl)-2Htetrazole (4)
A mixture of 1-(4-fluorophenyl)-1H-pyrazole-4-carbonitrile (9) (2.0 g, 12.4 mmol), sodium
azide (4.1 g,62 mmol), and ammonium chloride (3.35 g, 62 mmol) in 35 mL of DMF was heated at
reflux temperature for 72 h. The reaction mixture was then poured into water and acidified to pH 5.
The product was vacuum filtered and dried to provide 5-(1-(4-fluorophenyl)-1H-pyrazol-4-yl)-2H-
tetrazole (4) as a beige solid in a 98% yield; as Figure 2B shows, it was characterized as having mp >
227 °C, Rf = 0.82 (ethyl ether:EtOH, 7:3): IRmax (KBr) cm−1:3,135 (ν C–H), 1,635 (ν C=C), 1,212 (ν C–F),
1
838 (ν 1,4-C–F); H NMR (500.13 MHz) DMSO-d6 δ: 9.09 (1H, d, J = 0.5 Hz, H-5), 8.28 (1H, d, J = 0.5
Hz, H-3), 7.93 (1H, ddd, J = 9.1, 4.7, and 3.5 Hz,H-2′), 7.94 (1H, ddd, J= =.5, 4.7, and 3.5 Hz, H-6′), 7.38
(1H, ddd, J = 9.1, 8.5, and 3.5 Hz, H-3′), 7.38 (1H, ddd, J = 10.5, 9.1, and 3.5 Hz, H-5′); 13C NMR
(125.76 MHz) DMSO-d6 δ: 160.7 (C-4′), 149.3 (C-1”), 139.5 (C-3), 135.5 (C-1’), 127.8 (C-5), 121.1 (C-2’
and 6’), 116.7 (C-3’ and 5′), 108.6 (C-4), and [M+ H]+ of m/z 231.07854 (error = 1.5 ppm).
Antinociceptive activity
Acetic acid-induced abdominal writhing
Experimental groups of mice (n = 9) were treated orally (p.o.) with vehicle (10 mL/kg), FPPT
(9, 18, or 36 mg/kg p.o.), or indomethacin (10 mg/kg) 60 min prior to the administration of 1.2%
acetic acid solution (10 mL/kg, i.p.). The number of writhes produced in each group during the 30-
min test was counted and expressed as M ± SEM (23,24).
Formalin-induced pain
Experimental groups of mice (n = 8) were treated with vehicle (10 mL/kg, p.o.), FPPT (36
mg/kg p.o.), indomethacin (10 mg/kg, p.o. - positive control for antinociceptive activity in the second
phase) or morphine (5 mg/kg, s.c. - positive control for antinociceptive activity in the first and
second phases). Either 60 min after p.o. treatment or 30 min after s.c. treatment, 20 μL of 3%
formalin (in saline) was administrated into the plantar surface of the right hind paw. After being
injected with the phlogistic agent, the mice were placed in an acrylic box over a mirror to enable
unhindered observation of the formalin-injected paw for 30 min. Pain reaction time (i.e., licking
time) was assessed during two phases: 0–5 min for neurogenic pain and 15–30 min for inflammatory
pain, as described by Hunskaar and Hole (25). Results were expressed in seconds as M ± SEM.
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Tail flick test
Performed as described by D’Amour et al. (1941) (26), the tail flick test gauges time taken to
flick the tail (i.e., latency) during exposure to a heat source using an analgesimeter (Insight®, Ribeirão
Preto, Brazil). Animals were divided into three experimental groups (n = 10): the vehicle-treated
group (10 mL/kg, p.o.), the FPPT-treated group (36 mg/kg, p.o.), and the morphine-treated group
(5.0 mg/kg, s.c. - positive control for antinociceptive activity). Latent reaction to pain was measured
at -30, 0, 30, 60, 90, 120, 150, and 180 min of treatment with a cutoff set at 15 s. Results at all times
were expressed in seconds as M ± SEM.
Anti-inflammatory activity
Carrageenan-induced paw edema
Acute anti-inflammatory effect was evaluated by carrageenan-induced rat paw edema per
the method of Passos et al. (2007) (27). Experimental groups of mice (n = 9) were treated orally with
vehicle (10 mL/kg), FPPT (36 mg/kg), or indomethacin (10 mg/kg) 1 h before intraplantar injection of
carrageenan 1% in the right paw. The left paw was used as a control and received saline (NaCl 0.9%)
injection in the same volume. The edema was measured as the difference in the volume between
the paws using a plethysmometer (Ugo Basile Co., Monvalle, Italy) at intervals of 0, 1, 2, 3, and 4 h
following injection with the phlogistic agent.
Carrageenan-induced pleurisy
Groups of mice (n = 9) were treated orally with vehicle (10 mL/kg), FPPT (36 mg/kg), or
dexamethasone (2 mg/kg, p.o). Roughly 1 h after treatment, the animals received an injection of 100
μL 1% carrageenan into the pleural cavity, and 4 h after the phlogistic agent was administered, the
pleural exudate was collected with 1 mL of heparinized phosphate buffered saline (PBS). One aliquot
was used to count the total number of leukocytes using Türk’s solution in a Neubauer chamber
(28,29).
Preparation of rat aortic rings
Rats’ aortic rings free of fat and adherent connective tissues were cut into ring segments 4
mm long and suspended in 10-mL organ baths filled with modified Krebs–Henseleit solution (NaCl,
130 mM; NaHCO3, 14.9 Mm; KCl, 4.7 mM; KH2PO4, 1.18 mM; MgSO4⋅7H2O, 1.17 mM; CaCl2⋅2H2O, 1.6
mM; glucose, 5.5 mM at pH 7.4). The preparation was maintained with continuous supply of 5% CO2
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and 95% O2 mixture at 37 °C. The rings were passively stretched to a resting tension of 1.5 g and
allowed to achieve equilibrium for 60 min; the bath fluid was changed every 15 min. Alterations in
tension baseline were recorded by isometric transducers connected to a data acquisition system
(World Precision Instruments, Sarasota, FL, USA). Endothelia were removed mechanically by gently
rubbing a wet cotton swab through the luminal surface of the rings. Removed endothelia were
examined by vascular contraction and relaxation response to phenylephrine (PhE, 10−6 M) and
acetylcholine (ACH, 10−5 M), respecꢀvely (30).
Effects of FPPT on nitric oxide pathways, prostaglandins, and ion channels
The vascular activity of FPPT was assessed prior to the incubation of the endothelium-intact
rings with 100 μM L-NAME as the nitric oxide synthase inhibitor, 10 μM ODQ as the soluble
guanylylcyclase inhibitor, 10 μM indomethacin as the COX inhibitor, 1 mM TEA as the nonspecific
blocker of the voltage-dependent K+ channel, or 10 μM GB as the blocker of adenosine triphosphate
(ATP)-sensitive K+ channels. After an incubation period of 30 min, aortas were contracted with 0.1
μM PhE prior to the construction of a concentration-response curve to FPPT. Results were expressed
as percentage of the relaxation of the vascular contraction due to PhE. Endothelium-denuded rings
were then incubated in Ca2+-free high K+ (60 mM) depolarizing Krebs–Henseleit solution. A
cumulative concentration-response curve to CaCl2 (10–100 μM) was evaluated in the presence of
vehicle or FPPT. Results were expressed as percentages of the maximal response to KCl (120 mM).
Toxicological evaluation of FPPT
Cytotoxicity on BALB/c 3T3 cells
BALB/c 3T3-A31 fibroblasts were kindly donated by Dr. Mari Cleide Sogayar (Chemistry
Institute, São Paulo University, SP, Brazil). The cells were cultured in DMEM supplemented with 10%
fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin and routinely grown as a
monolayer in 75-cm2 tissue culture flasks in a humidified atmosphere of 5% CO2 in the air at 37 °C.
Cell cultures were removed from the flasks using trypsinization (trypsin:EDTA solution,
0.025%:0.02%) when cells exceeded 50% confluence but had not reached 80% confluence (31).
To evaluate the cytotoxicity of FPPT on 3T3 cells, the neutral red uptake assay was
performed as described by ICCVAM (2006) (32). Briefly, 3T3 fibroblasts (3 × 103 cells/well) were
seeded in 96-well plates and incubated overnight. Afterward, cells were treated with eight different
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concentrations of FPPT (0.01–1.4 mM) in complete medium and incubated for 48 h. Wells without
cells received the complete culture medium with or without FPPT. After the exposure period, the
supernatant was removed, and the cells were washed with 250 µL/well of prewarmed PBS followed
by the addition of 250 µL of NR medium in each well. After 3 h of incubation, the NR medium was
removed, the cells were carefully rinsed with 250 µL/well of prewarmed PBS, and 100 µL of NR
desorption solution (50 ethanol:1 acetic acid:49 ultrapure water) was added to each well.
Absorbance was measured at 550 nm. IC50 (mM) value—that is, the concentration that inhibited cell
growth by 50% compared to the untreated group—was used to estimate the LD50 (mg/kg), or the
lethal dose that causes death in 50% of animals, based on the equation of an established model
(31)—namely, log (LD50) = 0.545 × log (IC50) + 0.757.
Analysis of acute oral systemic toxicity in mice
The acute oral toxicity of FPPT was assessed by following OECD Guideline 423: Acute Toxic
Class Method (2001) (33). Briefly, male and female mice (n = 3 per sex) were treated orally by gavage
with a single dose (0.2 mL/mouse) of 2,000 mg/kg FPPT. Animals were observed during the first 10
min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h and daily thereafter through Day 14 post-treatment.
Hippocratic screening was performed to investigate the animals’ vocalizations, irritability, touch
response, response to tail tightening, righting reflex, body tone, tremors, convulsions, straub,
piloerection, cyanosis, salivation, and death. The intensities of the events were tabulated on a 5-
point scale (i.e., absent, minimal, little, moderate, and intense). The safety of LD50 obtained was
subsequently confirmed as recommended by OECD guidelines (33). The animals were euthanized 14
d after the experiments, at which point their abdominal cavities were opened to observe any
macroscopic alterations.
Statistical analysis
Results are expressed as M ± SE. Differences between two means were detected using
Student’s t test, whereas differences among more than two means were detected using one-way
analysis of variance (ANOVA) with a post hoc Student–Newman–Keuls’s test or two-way ANOVA with
Bonferroni’s post hoc test. Results were considered to be significant when p < .05 (34).
From data gathered during nociceptive and inflammatory tests, we calculated the
percentage of the effect of FPPT or positive control in relation to control group values. The mean
value of the control group was considered to be absolute, and differences in mean values were
identified in other groups as a percentage relative to control group’s mean value.
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Results
Synthesis of FPPT (4)
As illustrated in Figure 1B, the synthetic route began with 1-(4-fluorophenyl)-1H-pyrazole (5)
and proceeded through the classical method described by Finar and Godfrey (1954) (35).
Chemoselective and regiospecific formylation of 1-(4-fluorophenyl)-1H-pyrazole (7) to 1-(4-
fluorophenyl)-1Hpyrazole-4-carbaldehyde (8) was performed under Duff’s conditions (36). The
synthesis of 1-(4-fluorophenyl)-1H-pyrazole-4-carbonitrile (9) was conducted via oxime formation
with the reaction of 1-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde (8) with hydroxylamine,
followed by in situ dehydration in the presence of sodium iodide and DMF at reflux temperature for
6 h to produce (9) a 90% yield (37). FPPT (4) was synthetized through a 1,3-bipolar cycloaddition
between 1-(3-fluorophenyl)-1H-pyrazole-4-carbonitrile (9) and NaN3 using NH4Cl as the catalyst in
DMF at reflux temperature for 48 h in a 98% yield (38). FPPT (4) was obtained in a 55% overall yield.
Analgesic activity
Acetic acid-induced abdominal writhing
In the acetic acid-induced abdominal writhing test, FPPT (9, 18, or 36 mg/kg, p.o.) reduced
the number of writhes to 53.29 ± 4.8 in a dose-dependent manner (40% reduction, p < .001), 48.86 ±
1.6 (45% reduction, p < .001), and 35.86 ± 1.4 (60% reduction, p < .001), respectively (ANOVA: F(4–35)
= 33.34, p < .0001). The positive control indomethacin reduced the number of writhes to 51 ± 4.6
(42.6% reduction, p < .001) compared to the control group (number of writhes 88.9 ± 2.2) (Figure
3A).
Formalin-induced pain
In the formalin test, oral treatment with FPPT (36 mg/kg) showed antinociceptive activity
compared with the control group in the first (ANOVA: F(3,29) = 44.16, p < .0001) and second phases
(ANOVA: F(3,29) = 42.17, p < .0001) of the test. In the first phase, FPPT significantly reduced the licking
time to 52.21 ± 2.8 s (30.9% reduction, p < .01) compared with the control group (licking time 75.57
± 3.2 s), while in the second phase, the licking time was reduced to 76.29 ± 9.8 s (47% reduction, p <
.001) compared with the control group (licking time 144 ± 6.7 s). In the group treated with
morphine, licking time decreased significantly during both phases, to 6.63 ± 2.4 s (91.2% reduction, p
< .001) and 2.07 ± 2.1 s (98.6% reduction, p < .001), respectively. Treatment with indomethacin
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reduced only the second phase of the test to 83.22 ± 11.3 s (42.2% reduction, p < .001), as Figure 3B
shows.
Tail flick test
Tail flick test results revealed that oral treatment with FPPT (36 mg/kg) did not elicit any
significant antinociceptive activity compared to the group vehicle (two-way ANOVA, treatment
factor: F(2,210) = 191.9, p > .05). Under similar conditions, subcutaneous treatment with morphine (5
mg/kg, s.c. – opioid agonist) demonstrated significant antinociception at 30, 60, 90, 120, and 150
min post-treatment (data not shown).
Anti-inflammatory activity
Carrageenan-induced paw edema
The treatments with FPPT (36 mg/kg, p.o.) reduced the carrageenan-induced paw edema in
a dose-dependent manner throughout the test period (two-way ANOVA, treatment factor: F(2,135)
=
112.6, p < .0001). FPPT showed a significant anti-edematogenic effect by reducing the edema to 106
± 4.3 (19.7% reduction, p < .001), 106 ± 3.7 (25.9% reduction, p < .001), 102 ± 4.2 (25.5% reduction, p
< .001), and 91 ± 3.8 µL (27.2% reduction, p < .001) at 1, 2, 3 and 4 h, respectively, compared with
the control group (difference between paws: 132 ± 5.5, 143 ± 5.2, 137 ± 5.2, and 125 ± 2.7 µL,
respectively). The group treated with indomethacin (10 mg/kg, p.o.), an anti-inflammatory positive
control, exhibited decreased carrageenan-induced paw edema at 1, 2, 3, and 4 h post-treatment, as
Figure 4A illustrates.
Carrageenan-induced pleurisy
In the carrageenan-induced pleurisy test, treatment with FPPT at a dose of 36 mg/kg (p.o.)
reduced cell migration (ANOVA: F(2,29) = 36.15, p < .0001). The number of leukocytes × 106 /mL
dropped to 3.5 ± 0.3 (36.4% reduction, p < .001) compared with the group treated with the vehicle
(5.5 ± 0.3 leukocytes × 106/mL). The group treated with dexamethasone (2 mg/kg) also
demonstrated fewer leukocytes × 106 /mL to 1.9 ± 0.3 (65.5% reduction, p < .001) than the control
group (Figure 4B).
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Effects of FPPT on nitric oxide pathway, prostaglandin and ion channels
FPPT induced a concentration-dependent (1 µM–1 mM) vasodilation effect (maximal effect
[Emax]: 93.29 ± 2.05) in rats’ thoracic aortic rings with endothelia. The compound also induced a
concentration-dependent (1 µM–1 mM) relaxation effect (Emax: 91.86 ± 2.27) in endothelium-
denuded aortic rings (Figure 5), (two-way ANOVA, vessel factor: F(3, 131) = 129.8, p < .0001; FPPT
concentration factor: F(6, 131) = 126.1, p < .0001). As Figure 6A shows, the incubation of each aortic
ring with L-NAME or ODQ inhibited FPPT-induced vascular relaxation (two-way ANOVA, antagonist
factor: F(2, 105) = 66.56, p < .0001; FPPT concentration factor: F(6, 105) = 166.66, p < .0001).
Moreover, incubation of the aorta with TEA (Figure 6B) also inhibited FPPT’s relaxant effect (two-
way ANOVA, antagonist factor: F(1, 54) = 27.36, p < .0001; FPPT concentration factor: F(6, 54) =
85.38, p < .0001), as did GB in Figure 6C (two-way ANOVA, antagonist factor: F(1, 56) = 37.76, p <
.0001; FPPT concentration factor: F(6, 56) = 160.7, p < .0001) or indomethacin in Figure 6D (two-way
ANOVA, antagonist factor: F(1, 77) = 21.78, p < .0001; FPPT concentration factor: F(6, 77) = 162.6, p <
.0001). Contractile response to CaCl2 was blocked in a concentration-dependent manner by FPPT, as
Figure 7 illustrates (two-way ANOVA, FPPT concentration factor: F(3,160) = 55.77, p < .0001; CaCl2
concentration factor: F(9,160) = 69.02, p < .0001).
Evaluation of cytotoxicity on BALB/c 3T3 cells
After 48 h of exposure of FPPT, 3T3 cells showed less viability at high concentrations (0.35–
1.4 mM), yielding an IC50 value of 0.302 mM, from which LD50 was estimated in 685 mg/kg as the
initial dose of FPPT to be administered to mice for in vivo acute oral toxicity testing.
Analysis of acute oral sytemic toxicity in mice
Following exposure to a single FPPT dose of 2,000 mg/kg, both male and female mice
showed moderate body tremors and mild ptosis in the first 30 min. Until the second hour of
observation, the animals remained slightly lethargic, shrunken, and contracted in terms of body
tone, with slightly bristled hair. Afterward, no signs of toxicity were observed, nor was any mortality,
during the 14 d of study. Necropsy showed no macroscopic alterations in the organs of mice. As
such, FPPT was classified to be in Category 5 of the Harmonized Classification System for Chemical
Substances and Mixtures (GSH) and LD50 determined to be 2,000 mg/kg > LD50 < 5,000 mg/kg.
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Discussion
Previous reports have shown that pyrazole derivatives constitute an important class of
chemical compounds with a broad spectrum of biological activities, including analgesic, anti-
inflammatory (39,40), antipyretic (13), antimicrobial (11), vasorelaxant (16), antidepressant, and
anticonvulsant effects (12). With that knowledge, we performed classical in vivo and in vitro assays
to evaluate the new pyrazole derivative FPPT’s antinociceptive, anti-inflammatory, and vasorelaxant
effects, as well as the involvement of the NO/cGMP pathway and both K+ and Ca2+ channels in its
vasorelaxant effect. We also performed a toxicological analysis of FFPT.
The acetic acid-induced writhing test was performed in mice to evaluate any analgesic
effect. Local irritation provoked by intraperitoneal injection of acetic acid triggers the liberation of
various chemical mediators, including bradykinin, substance P, prostaglandins, and cytokines (e.g.,
IL-1β, TNF-α, and IL-8) (41). In that model, the oral administration of FPPT (9, 18, or 36 mg/kg)
reduced the number of writhes in a dose-dependent manner. Despite the satisfactory sensitivity of
the model to antinociceptive agents, the suppression of abdominal writhing response by muscle
relaxants, antihistamines, neuroleptics, and other drugs made acetic acid-induced writhing an
unspecific animal model of nociception (42).
To confirm the analgesic effect suggested in the writhing test, we also performed a formalin
test that identified two distinct phases of pain (43). The first phase, known as the neurogenic phase,
starts immediately after the intraplantar injection of formalin and is characterized by direct
stimulation of nociceptive fibers and the release of preformed mediators such as serotonin,
substance P, kinins, histamine, and calcitonin gene-related peptide (25,43,44), whereas the second
phase, or the persistent inflammatory phase, is associated with the formation or release of
inflammatory mediators such as cytokines, eicosanoids, and kinins (25,43,45). The time that animals
spent licking their paws (i.e., reactivity time to formalin-induced pain) in both phases of the test
decreased in the group of mice treated with FPPT in a dose of 36 mg/kg. According to Tjolsen et al.
(1992) (43), a distinct biphasic nociception induced by formalin allows that model of nociception to
advantageously discriminate central and peripheral components of pain. Shibata et al. (1989) (44)
have shown that central acting analgesics such as narcotics (e.g., morphine) can reduce both phases
of nociception, whereas peripherally acting drugs such as steroids (e.g., hydrocortisone and
dexamethasone) and non-steroidal anti-inflammatory drugs (e.g., indomethacin) primarily suppress
the late phase of nociception. However, data obtained regarding FPPT in the formalin test are not
sufficiently distinct to indicate whether their antinociceptive effect is centrally mediated.
To study centrally mediated antinociceptive action, we conducted the tail flick test, which
involves a spinal reflex, yet is subject to supraspinal influences. The reflex has been reported to be
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sensitive to opioid-like and analgesic drugs with central action (46,47). The effective dose of the
pyrazole derivative in the formalin test did not change the latency to thermal stimulation in the tail
flick test. The reference drug morphine, an opioid receptor agonist, induced a significant increase in
latency to thermal stimulation, as anticipated. Such results suggest that FPPT’s mechanism of
antinociceptive action does not involve central mechanisms.
To investigate the anti-inflammatory effect of FPPT, paw edema was induced via
carrageenan, a complex group of polysaccharides formed by repeating galactose-related monomers
(48). The development of paw edema in mice following carrageenan injection has been
characterized by multiple inflammatory mediators and exudate of inflammation (49). The first 2 h
after carrageenan injection predominantly has been shown to involve the release of
proinflammatory agents such as histamine, serotonin, and bradykinin, while subsequent hours can
entail the release of prostaglandins (50). In our study, FPPT and indomethacin significantly reduced
the carrageenan-induced paw edema throughout the test period.
We performed the carrageenan-induced pleurisy test to confirm the anti-inflammatory
effect of FPPT. This acute inflammation model is characterized by the accumulation of exudate and a
rapid influx of polymorphonuclear cells, followed by mononuclear cell infiltration in the inflamed
pleural cavity, thereby allowing the quantification of migrated cells (28,51,52). In that test,
treatment with FPPT at a dose of 36 mg/kg significantly reduced cell migration, thereby confirming
the anti-inflammatory effect of FPPT in the second phase of the formalin-induced pain and the
carrageenan-induced paw edema tests.
Our results showed an endothelium-independent vasorelaxant effect of FPPT in the isolated
organ model, as consistent with an earlier study on LQFM021 (16), an analog of FPPT. The vascular
relaxation effect of FPPT, which showed that the compound is a vasodilator, excludes a false positive
effect on the redution of edema and cell migration that could be associated with a vasoconstrictive
property.
Nitric oxide (NO) is involved in multiple cellular functions and recognized as a major
physiological regulator of vascular tone (53,54). NO is also the chief activator of soluble guanylate
cyclase, an enzyme that synthesizes guanosine-3-5-cyclic monophosphate, the level of which is
regulated by specific phosphodiesterases (PDE). Several PDE inhibitors have been developed for use
as therapeutic agents since they increase cyclic nucleotide levels by blocking PDE function to
enhance NO-cGMP signalization (55).
To evaluate the involvement of NO in FPPT’s vasorelaxant effect, isolated aortic rings were
incubated with L-NAME—that is, the inhibitor of endothelial nitric oxide synthase—or ODQ, a
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guanylate cyclase inhibitor. Both L-NAME and ODQ blocked the effect of FPPT, thereby suggesting
the involvement of the NO/cGMP pathway.
To investigate the involvement of K+ channels on vasorelaxant effect of FPPT, aortic rings
were incubated with TEA, a nonselective inhibition of voltage-dependent K+ channels. Incubation
with TEA blocked FPPT’s vasorelaxant effect, while incubation with glibenclamide, a blocker of ATP-
sensitive K+ channels, attenuated the compound’s vasorelaxant effect, thereby showing the
involvement of K+ channels on its vasorelaxant effect.
Studies have also shown that the activation of the L-arginine-NO-cGMP pathway K+ channel
is also involved in the antinociceptive effect of certain drugs. L-arginine is a substrate that NOS uses
to produce NO, which subsequently activates guanylate cyclase, thereby stimulating the synthesis of
cGMP that can interact with K+ channels and promote its opening. The opening of K+ channels leads
to the hyperpolarization of the cell membrane and prevents the generation of nociceptive stimuli
(56,57). It has been reported that the blockade of K+ channels can moreover reduce the analgesic
effect (18,58).
As the literature shows, several drugs used to treat pain or inflammation have exhibited the
participation of NO/cGMP, prostanoids, and ionic channels in their mechanisms of action (17,59).
Dipyrone, diclofenac, ketorolac, ketamine, and morphine are classic examples of analgesics that act
via the NO/cGMP pathway (18,60,61), and several preclinical studies seeking new substances with
antinociceptive and anti-inflammatory activities have revealed the involvement of NO/cGMP and K+
channels (62–64).
Considering that LQFM021, an analog of FPPT, was able to inhibit PDE3 and produce
vasorelaxant effects similar to the tested compound in a docking assay (16), as well as showed an
antinociceptive effect in the first phase of the formalin test, an effect blocked by NO/cGMP/K+
channel pathway inhibitors (15), results for FPPT in the formalin test and isolated aortic rings assay
support the hypothesis that the antinociceptive effect of FPPT involves those pathways, along with
its anti-inflammatory effect demonstrated during edema and pleurisy tests.
Other than NO, vasoactive substances such as prostanoids are important modulators of
vascular tone. Their production is directly related to the availability of arachidonic acid substrate and
enzyme cyclooxygenases. Prostacyclin (PGI2) is a member of the prostanoids group that in blood
vessels performs an important vasodilatory function that overrides the vasoconstrictive function of
thromboxane A2 (65,66). Therefore, to evaluate the involvement of prostanoids in FPPT’s vascular
effect, aortic rings were incubated with indomethacin, a known cyclooxygenase inhibitor. The
rightward shift of the dose-effect curve demonstrates the blockade of FPPT due to indomethacin,
which, as a result, requires additional study to ascertain the involvement of COX in pyrazole
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derivative’s biological activities. At the same time, its effect is highly similar to that of other pyrazole
derivatives reported in the literature (16,67).
Other findings in our study demonstrate that FTTP can alter the contractile effect of a
cumulative addition of CaCl2 in aortic rings kept under depolarising conditions. That result suggests
the involvement of voltage-sensitive Ca2+ channels at the plasmatic membrane. The blockade of
voltage-dependent Ca2+ channels have been implicated in the mechnisms of antinociceptive and
anti-inflammatory actions of some drugs (20,68,69). FPPT attenuated the contractile effect of a
cumulative addition of CaCl2 in the aorta maintained in depolarizing conditions. The attenuation of
the contractile effect indicates that FPPT’s vasorelaxant effect could involve a blockade of Ca2+
channels independent of K+ channels or NO, if not both.
In higher concentrations (0.35–1.4 mM), FPPT promoted the reduction of 3T3 basal cells’
viability. The acute oral toxicity of FPPT was assessed according to OECD Guideline 423: Acute Toxic
Class Method (2001), a method that uses predefined doses and the results of which allow a
substance to be ranked and classified according to the Globally Harmonized System for the
classification of chemicals that cause acute toxicity. The principle of the test is that, based on a
stepwise procedure using a minimum number of animals per step, sufficient information can be
obtained about the acute toxicity of the tested substance to enable its classification (33). In that
sense, FPPT was orally tolerated well in a dose of 2,000 mg/kg despite some signals of toxicity 2 h
after exposure; it can thus be classified at GSH Category 5, which includes substances with a low
acute toxicity hazard. Our findings are similar to those of its synthetic analog (16).
Conclusion
In all, the new pyrazole derivative FPPT showed a low toxicological profile according to OECD
Guideline 423: Acute Toxic Class Method and was classified in Category 5 of the Harmonized
Classification System for Chemical Substances and Mixtures, with LD50 determined to be 2,000
mg/kg > LD50 < 5,000 mg/kg. The antinociceptive effect of the compound was observed in an acetic
acid-induced abdominal writhing test and in a formalin-induced pain test. FPPT also reduced the
edema formation and cell migration in a pleurisy test, thereby confirming its anti-inflammatory
effect. It furthermore demonstrated a vasorelaxant effect, whose reduction by indomethacin, L
NAME, ODQ, TEA, and GB suggests the involvement of NO/cGMP and K+ channels in the effect. That
pathway might also modulate the antinociceptive effect, while the blockade of voltage-dependent
calcium channels could have contributed to the vasorelaxation effect.
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From the perspective of developing new drugs, additional pharmacological tests that
evaluate the role of NO in the anti-inflammatory effect of FPPT should be conducted, as well as
studies that evaluate the hypotensive or antihypertensive effect of FPPT in rats. Performing such
preclinical toxicity tests can better ensure the safe pharmacology of this new compound.
Acknowledgments
The authors are grateful to Dra. Ekaterina A. F. B. Rivera and Lucas B. do Nascimento for
ethical and technical assistance, as well as to the Conselho Nacional de Desenvolvimento Científico e
Tecnológico and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for financial
support.
Conflict of interest statement
The authors declare no conflict of interest.
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Figure Legends
Figure 1. Structural design and synthetic route. (A) Structural design concept of new 5-(1-(4-
fluorophenyl)-1H-pyrazol-4-yl)-1H-tetrazole (4) and (B) synthetic route for the preparation of 5-(1-
(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-tetrazole (4).
Figure 2. Analytical data. (A) ESI (+) FT-Orbitrap MS spectra obtained for 1-(4-fluorophenyl)-1H-
pyrazole-4-carbonitrile. (B) ESI (+) FT-Orbitrap MS spectra obtained for 5-(1-(4-fluorophenyl)-1H-
pyrazol-4-yl)-2H-tetrazole.
Figure 3. Antinociceptive effect of FPPT. (A) The animals treated with FPPT at doses 9, 18 or 36
mg/kg p.o., reduced significantly the number of acetic acid-induced writhing in mice (n= 9) in dose-
dependent manner. Indomethacin (10 mg/kg p.o.) was used as a positive control. (ANOVA: F(4-35)
=
33.34, P < 0.0001). (B) FPPT at dose 36 mg/kg p.o., reduced significantly the licking time (s) in the
first phase and second phase of formalin test in mice (n= 8). Indomethacin (10 mg/kg, p.o.) and
morphine (5 mg/kg, s.c.) were used as positive controls. [First phase (ANOVA: F(3,29) = 44.16, P <
0.0001) and the second phase (ANOVA: F(3,29) = 42.17, P < 0.0001) of the test]. This results are
presented as mean ± S.E.M. for each experimental group. **P< 0.01; ***P < 0.001 compared with
control group treated with vehicle (10 mL/kg p.o.), acoording to ANOVA followed by Student-
Newman-Keuls’ test.
Figure 4. Anti-inflammatory effect of FPPT. (A)The treatmet with FPPT (36 mg/kg p.o.) reduced
significantly the edema induced by carrageenan in mice (n = 10) in all hours of test. Indomethacin
(10 mg/kg p.o.) was used as positive control. The results are presented as mean ± S.E.M. of the
difference between the paws, in μL. ***P < 0.001 compared with control group, acoording to two-
way ANOVA followed by Bonferroni's test. (Two-way ANOVA, treatment factor: F(2,135) = 112.6, P <
0.0001). (B) FPPT at dose 36 mg/kg p.o. also reduced significantly the number of leukocytes migrated
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for the pleural cavity of mices (n = 10) in carrageenan-induced pleurisy test. Dexamethasone (2
mg/kg p.o.) was used as positive control. The results are presented as mean ± S.E.M. of the number
of leukocytes migrated. ***P < 0.001 compared with control group, acoording to ANOVA followed
by Student-Newman-Keuls’ test (ANOVA: F(2,29) = 36.15, P < 0.0001).
Figure 5. Vasorelaxant effect of compound FPPT in thoracic aorta of rats (n=6). Cumulative
concentration-response curves for FPPT (1 µM to 1 mM) in rats aortic precontracted with
phenylrphrine (0.1 µM) in endothelium-intact aortic rings (E+) or endothelium-denuded (E−) aorꢀc
rings. **P< 0.01; ***P < 0.001 compared with control group, acoording to two-way ANOVA followed
by Bonferroni's test. (Two-way ANOVA, vessel factor: F(3, 131)= 129.8, P < 0.0001; FPPT concentration
factor: F(6, 131)= 126.1, P < 0.0001).
Figure 6. Cumulative concentration-response curves for FPPT (1 µM to 1 mM) in rats aortic rings
(n=6) with intact endothelium precontracted with phenylrphrine (0.1 µM) in the presence of (A) Nω-
nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase inhibitor, 100 µM or 1H-
[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) a guanylylcyclase inhibitor, 100 µM (Two-way
ANOVA, antagonists factor: F(2, 105)=66.56, P < 0.0001; FPPT concentration factor: F(6, 105)=166.66, P <
0.0001); (B) Tetraethylammonium (TEA) voltage-dependent K+ specific channel blocker, 1 µM (Two-
way ANOVA, antagonists factor: F(1, 54)=27.36, P < 0.0001; FPPT concentration factor: F(6, 54)=85.38, P
< 0.0001); (C) Glibenclamide (GB) a seletive inhibitor of channels KATP, 100 µM (Two-way ANOVA,
antagonists factor: F(1, 56)=37.76, P < 0.0001; FPPT concentration factor: F(6, 56)=160.7, P < 0.0001); or
(D) Indomethacin (INDO) a cyclooxygenase inhibitor, 10 µM (Two-way ANOVA, antagonists factor: F(1,
77)=21.78, P < 0.0001; FPPT concentration factor: F(6, 77)=162.6, P < 0.0001). **P< 0.01; ***P < 0.001
compared with control group, acoording to two-way ANOVA followed by Bonferroni's test.
Figure 7. Effect of FPPT (0.1, 0.3 and 1 mM) on the contractile responses elicited by incremental
addition of Ca2+ (10 µM to 0.1 mM) to aortic rings depolarized by KCl (60 mM) in a Ca2+-free solution.
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Contractile responses are expressed as the percentage of maximum contraction evoked by KCl (120
mM) (n=6). Contractile responses are expressed as the percentage of maximum contraction evoked
by KCl (120 mM) (n=6). **P<0.01; ***P < 0.001 compared with control group, acoording to two-way
ANOVA followed by Bonferroni's test. (Two-way ANOVA, FPPT concentration factor: F(3,160)=55.77, P
< 0.0001; CaCl2 concentration factor: F(9,160)=69.02, P < 0.0001).
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

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