Molecular docking and pharmacological/toxicological assessment of a new compound designed from celecoxib and paracetamol by molecular hybridization

Article abstract of DOI:10.1007/s10787-018-0516-7

Nonsteroidal anti-inflammatory drugs are commonly used worldwide; however, they have several adverse effects, evidencing the need for the development of new, more effective and safe anti-inflammatory and analgesic drugs. This research aimed to design, syn

Full text of DOI:10.1007/s10787-018-0516-7

Infammopharmacology
https://doi.org/10.1007/s10787-018-0516-7
Inflammopharmacology
ORIGINAL ARTICLE
Molecular docking and pharmacological/toxicological assessment
of a new compound designed from celecoxib and paracetamol
by molecular hybridization
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Daiany P. B. da Silva · Iziara F. Florentino · Dayane M. da Silva · Roberta C. Lino · Carina S. Cardoso ·
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Lorrane K. S. Moreira · Géssica A. Vasconcelos · Daniela C. Vinhal · Anna C. D. Cardoso · Bianca Villavicencio ·
Hugo Verli · Boniek G. Vaz · Luciano M. Lião · Luiz C. da Cunha · Ricardo Menegatti · Elson A. Costa
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Received: 3 April 2018 / Accepted: 9 July 2018
©
Springer Nature Switzerland AG 2018
Abstract
Nonsteroidal anti-infammatory drugs are commonly used worldwide; however, they have several adverse eꢀects, evidencing
the need ꢁor the development oꢁ new, more eꢀective and saꢁe anti-infammatory and analgesic drugs. This research aimed
to design, synthesize and carry out a pharmacological/toxicological investigation oꢁ LQFM-102, which was designed ꢁrom
celecoxib and paracetamol by molecular hybridization. To evaluate the analgesic eꢀect oꢁ this compound, we perꢁormed
ꢁ
ormalin-induced pain, hot plate and tail fick tests. The anti-infammatory eꢀect oꢁ LQFM-102 was evaluated in carrageenan-
induced paw oedema and pleurisy tests. The biochemical markers indicative oꢁ toxicity—AST, ALT, GSH, urea and creati-
nine—as well as the index oꢁ gastric lesion aꢁter prolonged administration oꢁ LQFM-102 were also analyzed. In addition, the
interaction oꢁ LQFM-102 with COX enzymes was evaluated by molecular docking. In all experimental protocols, celecoxib
or paracetamol was used as a positive control at equimolar doses to LQFM-102. LQFM-102 reduced the pain induced by
ꢁormalin in both phases oꢁ the test. However, this compound did not increase the latency to thermal stimuli in the hot plate
and tail fick tests, suggesting an involvement oꢁ peripheral mechanisms in this eꢀect. Furthermore, LQFM-102 reduced paw
oedema, the number oꢁ polymorphonuclear cells, myeloperoxidase activity and TNF-α and IL-1β levels. Another interesting
ꢂnding was the absence oꢁ alterations in the markers oꢁ hepatic and renal toxicity or lesions oꢁ gastric mucosa. In molecular
docking simulations, LQFM-102 interacted with the key residues ꢁor activity and potency oꢁ cyclooxygenase enzymes, sug-
gesting an inhibition oꢁ the activity oꢁ these enzymes.
Keywords Gastric toxicity · Hepatotoxicity · Cytokines · Celecoxib · Paracetamol · LQFM-102
Introduction
Despite the notorious adverse eꢀects mainly related to gas-
trointestinal tract and renal dysꢁunction, the nonsteroidal
anti-infammatory drugs (NSAIDs) constitute a class oꢁ
medications most commonly used worldwide (Sostres
Electronic supplementary material The online version oꢁ this
article (https://doi.org/10.1007/s10787-018-0516-7) contains
supplementary material, which is available to authorized users.
3
*
Elson A. Costa
xico@uꢁg.br
Nucleus oꢁ Studies and Research Toxicopharmacological,
Federal University oꢁ Goiás, Goiânia, GO, Brazil
4
Laboratory oꢁ Medicinal Pharmaceutical Chemistry,
1
Laboratory oꢁ Pharmacology oꢁ Natural and Synthetic
Products, Department oꢁ Pharmacology, Institute
oꢁ Biological Sciences, Federal University oꢁ Goiás,
Campus Samambaia - ICB 2 - Sala 216, CP 131, Goiânia,
GO CEP 74001-970, Brazil
Faculty oꢁ Pharmacy, Federal University oꢁ Goiás, Campus
Samambaia, Goiânia, GO, Brazil
5
Center oꢁ Biotechnology, Federal University oꢁ Rio Grande
do Sul, Porto Alegre, RS, Brazil
2
Chemistry Institute, Federal University oꢁ Goiás, Campus
Samambaia, Goiânia, GO, Brazil
Vol.:(0
1
123456789)
3

D. P. B. da Silva et al.
et al. 2013; Ungprasert et al. 2012). Traditional NSAIDs
inhibit both COX isoꢁorms, while speciꢂc COX-2 inhibi-
tors, known as coxibs, have a higher speciꢂcity ꢁor the
COX-2 isoꢁorm, thus reducing the adverse eꢀects associ-
ated with the inhibition oꢁ COX-1 isoꢁorm (Ungprasert
et al. 2012). A saꢁety proꢂle in superior gastrointestinal
tract related to the use oꢁ speciꢂc COX-2 inhibitors has
been demonstrated in several randomized controlled trials
and epidemiological studies (Bensen et al. 2000; Mamdani
et al. 2002; McKenna et al. 2001; Silverstein et al. 2000);
however, these inhibitors increased the risk oꢁ serious
cardiovascular events (Bresalier et al. 2005; McGettigan
and Henry 2006). Moreover, COX-2-selective NSAIDs
are also capable oꢁ developing adverse eꢀects related to
the renal system, similar to those caused by conventional
NSAIDs (Kummer and Coelho 2002). Currently, the only
coxib approved ꢁor clinical use in the USA is celecoxib
Based on this background, the present study aimed at the
development and pharmacological assessment oꢁ LQFM-
102 (3) compound, a heterocyclic derivative, which was
designed by molecular hybridization strategy ꢁrom the anti-
infammatory celecoxib (1) and the analgesic paracetamol
(2) (Fig. 1a).
Materials and methods
Analytical instruments
Reactions were monitored by TLC using commercially
available precoated plates (Whatman 60 F254 silica), and
developed plates were examined under UV light (254
1
13
and 365 nm). H and C NMR spectra were recorded in
indicated solvents on a Bruker Avance III 500 MHz spec-
trometer. Chemical shiꢁts are reported in parts per million
downꢂeld ꢁrom TMS and the coupling constants are in
(
Burnier 2005). However, Etoricoxib, Parecoxib and other
COX-2 selective drugs have been marketed in other coun-
tries worldwide (Reuben 2009).
1
13
Hertz. All signal assignments in H and C NMR spectra
are consistent with the chemical structures oꢁ the products
described. Inꢁrared spectra were recorded on a Perkin–Elmer
Spectrum Bx-II FT-IR System spectrophotometer as ꢂlms
on KBr discs. Melting points were determined using a
Marte melting point apparatus and the results were uncor-
rected. Mass spectra (MS) were obtained with a Q-Exactive
(Thermo Scieniꢂc, Bremen, Germany). The sample prepa-
ration ꢁor mass spectrometry analysis consisted oꢁ diluting
0.5 mg oꢁ each sample in 1 mL oꢁ methanol. To perꢁorm the
analysis in positive mode, 1 μL oꢁ ꢁormic acid was added
to the samples. The solution obtained was directly inꢁused
at a fow rate oꢁ 5 μL/min into the ESI source. The ESI (±)
source conditions were as ꢁollows: a nebulizer gas pressure
oꢁ 0.5–1.0 bar, a capillary voltage oꢁ 3.5 kV and a transꢁer
capillary temperature oꢁ 275 °C. The organic solutions were
dried over anhydrous sodium sulꢁate and then distilled under
reduced pressure in a rotary evaporator.
One oꢁ the traditional NSAIDs more commonly used
worldwide due to its analgesic and antipyretic eꢀects is par-
acetamol; however, its analgesic action mechanism is still
poorly understood (Moriarty and Carroll 2016). Moreover,
this drug exhibits low anti-infammatory activity in thera-
peutic doses (Graham et al. 2013; Whitehouse and Butters
2
014) and is the predominant reason ꢁor acute liver ꢁailure
(
ALF) in industrialized countries, including United States,
where it accounts ꢁor more than 50% oꢁ overdose-related
acute liver ꢁailures and approximately 20% oꢁ the liver trans-
plant cases (Brune et al. 2015; Yoon et al. 2016).
In this context, the design, synthesis and development
oꢁ new compounds by diꢀerent strategies to optimize the
pharmacokinetic and pharmacodynamic parameters oꢁ
known compounds emerge as an alternative to the develop-
ment oꢁ more eꢀective, low-toxicity lead compounds (Wer-
muth 2003). For example, based on the discovery oꢁ the
class oꢁ pyrazole compounds, which combined dipyrone and
celecoxib, ꢁor instance, it has been possible to develop sev-
eral agents with a wide range oꢁ pharmacological activities,
such as antipyretic (Souza et al. 2002), analgesic (Milano
et al. 2008; Oliveira et al. 2016) and anti-infammatory
Syntheses
Synthesis of methyl
1‑(4‑aminophenyl)‑1H‑pyrazole‑4‑carboxylate (9) (Petrini
et al. 1987)
(
Cidade et al. 2016; Ragab et al. 2013) eꢀects.
Previous studies have reported the antinociceptive and
anti-infammatory eꢀects oꢁ pyrazole compounds in diꢁ-
To a stirred heterogeneous mixture oꢁ methyl
1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate (8) (247 mg,
1.0 mmol) in 4 mL MeOH, 10% palladium-on-carbon
(40 mg) was added. The mixture was cooled in an ice
ꢁ
erent experimental models in vivo, using low doses and
presenting low toxicity (Amir et al. 2008; Florentino et al.
016; Gokhan-Kelekc et al. 2007; Oliveira et al. 2016; Rani
2
et al. 2004; Rathish et al. 2008). Moreover, some pyrazole
compounds have shown an ability to inhibit the synthesis oꢁ
pro-infammatory cytokines (Keche et al. 2012 and Townes
et al. 2004), becoming an interesting source ꢁor novel anal-
gesic and anti-infammatory drugs.
bath, and NaBH (94 mg, 2.5 mmol) was added in three
4
portions over 10 min. The reaction mixture was heated at
refux temperature ꢁor 1 h and then partitioned between
50 mL each oꢁ 2 N HCl solution and CHCl , and the solid
3
1
3

Molecular docking and pharmacological/toxicological assessment of a new compound designed…
Fig. 1 a Structural design oꢁ LQFM-102 (3) ꢁrom celecoxib (1) and paracetamol (2). b Synthetic route ꢁor the preparation oꢁ
1
-(4-acetamidophenyl)-1H-pyrazole-4-carboxylic acid-LQFM-102 (3)
was ꢂltered oꢀ and washed with 2 × 10 ml oꢁ water. The
phases were separated and the aqueous layer was extracted
Synthesis of methyl
1‑(4‑acetamidophenyl)‑1H‑pyrazole‑4‑carboxylate (10)
(Borah et al. 1997)
3
times with 15 mL CH Cl , and the combined organic
2 2
layers were dried over Na SO and concentrated in vacuo,
2
4
and the crude product was puriꢂed by column chroma-
To a stirred solution oꢁ methyl 1-(4-aminophenyl)-
1H-pyrazole-4-carboxylate (9) (217 mg, 1.0 mmol) in
acetic anhydride (0.094 mL, 1.05 mmol) was added iodine
(12.6 mg, 0.1 mmol) at room temperature ꢁor 30 min.
Then, the residue was partitioned between saturated
NaHCO solution and CH Cl . The phases were separated
tography (SiO , hexane/EtOAc = 5:5) to aꢀord methyl
2
1
-(4-aminophenyl)-1H-pyrazole-4-carboxylate (9) (97 mg,
4
5%) as a beige solid, Rf = 0.37 (CH Cl /MeOH, 90:10),
2
2
−
1
MP = 160-163 °C; IR
(KBr) cm : v 3434 and 3236
max
(
(
NH ), v 3113 (C–H aromatic), v 1702 (C = O) and v 825
2
3
2
2
1
1,4-aromatic); H NMR [500.13 MHz, J (Hz)] DMSOd-
and the aqueous layer was extracted 3 times with 15 mL
6
δ: 8.86 (1H, d, J = 0.6 Hz, H-5); 8.10 (1H, d, J = 0.6 Hz,
H-3); 7.59 (2H, d, J = 8.9 Hz, H-2′ and 6′); 6.73 (2H, d,
J = 8.9 Hz, H-3′ and 5′); 5.56 (2H, s, H-8); 3.84 (3H, s,
CH Cl , and the combined organic layers were dried over
2
2
Na SO and concentrated in vacuo, and the crude product
2
4
was puriꢂed by column chromatography (SiO , hexane/
2
1
3
H-7); C NMR (125.76 MHz) DMSOd- δ: 162.7 (C-6);
EtOAc = 3:7) to aꢀord methyl 1-(4-acetamidophenyl)-
1H-pyrazole-4-carboxylate (10) (114 mg, 44%) as a white
solid, Rf=0.50 (CH Cl /MeOH, 90:10) MP=216–219 °C;
6
1
48.4 (C-4′); 140.6 (C-3); 129.8 (C-5); 128.9 (C-1′); 120.5
(
(
C-2′ and 6′); 114.8 (C-4); 113.6 (C-3′ and C-5′); 50.9
2
2
+
−1
C-7); MS: [M + H] m/z oꢁ 218.09232 (Supplementary
IR (KBr) cm : v 3282 (N–H), v 3122 (C–H aromatic),
max
data).
v 1724 (C=O ester), v 1663 (C=O amide) and v 827
1
(
1,4-aromatic); H NMR [500.13 MHz, J (Hz)] DMSOd-
6
1
3

D. P. B. da Silva et al.
δ: 10.20 (1H, s, H-8); 9.07 (1H, d, J = 0.5 Hz, H-5); 8.19
Drugs and chemicals
(
1H, d, J = 0.5 Hz, H-3); 7.91 (2H, d, J = 9.1 Hz, H-3′ and
5
2
1
′); 7.78 (2H, d, J=9.1 Hz, H-2′ and 6′); 3.87 (3H, s, H-7);
The chemicals used in this study were paracetamol
1
3
®
.14 (3H, s, H-10); C NMR (125.76 MHz) DMSOd- δ:
(Paracetamol , Medley, SP, Brazil), acetone (Synth, Dia-
6
68.7 (C-9); 162.4 (C-6); 141.5 (C-3); 138.6 (C-4′); 134.1
dema, SP, Brazil), carrageenan (Sigma Chemicals, USA),
®
(
C-1′); 130.7 (C-5); 119.6 (C-3′ and 5′); 119.4 (C-2′ and
celecoxib (Celebra , Pꢂzer, SP, Brazil), croton oil (Sigma
+
6
′); 115.6 (C-4); 51.3 (C-7); 23.8 (C-10); MS: [M + H]
Aldrich, USA), dimethyl sulꢁoxide (DMSO, Sigma Chemi-
cals, USA), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB,
Sigma Chemicals, USA), ꢁormaldehyde (Synth, Diadema,
SP, Brazil), heparin (Hipolabor, Belo Horizonte, MG, Bra-
zil), IL-1β Elisa Ready-Set-GO Mouse Kit (eBioscience,
m/z oꢁ 260.10284 (Supplementary data).
Synthesis
®
of 1‑(4‑acetamidophenyl)‑1H‑pyrazole‑4‑carboxylic acid (3)
nº 88–7013), morphine hydrochloride (Dimorꢁ , Cristalia,
(Khurana et al. 2004)
SP, Brazil), o-dianisidine (Sigma Chemicals, USA), sodium
azide (Sigma Chemicals, USA), TNF-alpha Elisa Ready-Set-
GO Mouse Kit (eBioscience, nº 88-7324), trichloroacetic
acid (TCA, Synth, Diadema, SP, Brazil), and Türk solution
(New Prov, Pinhais, PR, Brazil). Colorimetric COX (ovine)
inhibitor screening assay (nº 760111), and lipoxygenase
(soybean) inhibitor screening assay (nº 760700) kits were
obtained ꢁrom Cayman Chemical Company.
To a stirred solution oꢁ potassium hydroxide (1358.9 mg,
4.2 mmol) in THF:MeOH:H O (5.8:2.0:2.0 L) was added
2
2
methyl 1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate (8)
(
259 mg, 1.0 mmol) at room temperature ꢁor 2 h. The
reaction mixture was then poured into water and acidi-
ꢂed to pH 4, and the product was vacuum ꢂltered and
dried. The crude product was puriꢂed by column chro-
matography (SiO , hexane/EtOAc = 0.5:9.5) to aꢁꢁord
Formalin test
2
1
-(4-acetamidophenyl)-1H-pyrazole-4-carboxylic acid (3)
(
164 mg, 67%) as a white solid, Rf=0.03 (CH Cl /MeOH,
The ꢁormalin-induced nociception test was perꢁormed as
described previously by Hunskaar et al. (1985). Mice (n=8)
were treated with vehicle (10% DMSO, 10 mL/kg, p.o.),
LQFM-102 (3) (0.3, 0.6 and 1.2 mmol/kg, p.o.), paracetamol
(2) (0.6 mmol/kg, p.o.) or celecoxib (1) (0.6 mmol/kg, p.o.).
Sixty minutes aꢁter the treatment, 20 μL oꢁ 3% ꢁormalin was
injected into the plantar surꢁace oꢁ the right hind paw. Aꢁter
that, the mice were placed in an acrylic box and a mirror was
placed under the box to enable unhindered observation oꢁ the
ꢁormalin-injected paw ꢁor 30 min. Pain reaction time (licking
time) was assessed during two periods: 0–5 min, the ꢂrst
phase, when the neurogenic pain is caused by direct stimula-
tion oꢁ the nociceptors, and ꢁrom 15 to 30 min, the second
phase, when the infammatory pain is caused by the release
oꢁ infammatory mediators. These results were expressed as
the mean± SEM oꢁ licking time in seconds (s).
2
2
−
1
9
0:10) MP > 250 °C; IR
(KBr) cm : v 3500-2500
max
(
O–H), v 3296 (N–H), v 1672 (C=O acid), v 1660 (C=O
1
amide) and v 829 (1,4-aromatic); H NMR [500.13 MHz,
J (Hz)] DMSOd- δ: 10.24 (1H, s, H-8); 8.97 (1H, brs,
6
H-5); 8.12 (1H, brs, H-3); 7.90 (2H, d, J = 8.9 Hz, H-3′
and 5′); 7.79 (2H, d, J = 8.9 Hz, H-2′ and 6′); 2.14 (3H, s,
1
3
H-10); C NMR (125.76 MHz) DMSOd- δ: 168.0 (C-9);
6
1
64.3 (C-6); 142.3 (C-3); 138.5 (C-4′); 134.7 (C-1′); 131.2
(
(
C-5); 119.8 (C-3′ and 5′); 119.8 (C-2′ and 6′); 117.5
+
C-4); 24.4 (C-10); MS: [M + H] m/z oꢁ 246.08719 (Sup-
plementary data).
Pharmacological evaluation
Animals
Tail ꢀick test
Experiments were perꢁormed using ꢁemale Swiss albino
mice (25–30 g) ꢁrom the Central Animal House oꢁ the Uni-
versidade Federal de Goiás (UFG). The animals were kept
in plastic cages at 22± 2 °C, with ꢁree access to pellet ꢁood
and water and under a 12 h light/dark cycle, in compliance
with the International Guiding Principles ꢁor Biomedical
Research Involving Animals. The animals were acclima-
tized ꢁor 7 days beꢁore the beginning oꢁ the experiments.
All experimental protocols were developed and approved
according to the principles oꢁ ethics and animal welꢁare des-
ignated by the Ethics Committee on Animal Use oꢁ UFG
The tail fick test was perꢁormed as described previously by
D`amour and Smith (1941). In this test, the time taken to
fick the tail (latency) when it was exposed to a heat source
®
was measured using an analgesimeter (Insight , Ribeirão
Preto, São Paulo, Brazil). The animals were divided into
ꢁour experimental groups (n = 8): vehicle (10% DMSO,
10 mL/kg, p.o.), LQFM-102 (3) (0.6 mmol/kg, p.o.), par-
acetamol (2) (0.6 mmol/kg, p.o.) or morphine (0.017 mmol/
kg, s.c.—positive control ꢁor antinociceptive activity). The
latency baseline was perꢁormed 30 min beꢁore the treatments
(time − 30) and immediately aꢁter oꢁ the treatments (time
(
number: 182/10).
1
3

Molecular docking and pharmacological/toxicological assessment of a new compound designed…
0
) ꢁor each animal. Aꢁter that, the latency to pain reaction
content using Türk’s liquid in a Neubauer’s chamber and the
other aliquot was used ꢁor the diꢀerential count oꢁ leukocyte
mononuclear and polymorphonuclear cells. The results oꢁ
the total and diꢀerential counts were expressed as the abso-
lute numbers oꢁ total cells and each type oꢁ cell, respectively,
which was oꢁten more inꢁormative than their proportion. The
ꢂnal result was also adjusted according to the volume unit
used in the perꢁormed evaluation and was expressed as the
was measured at 30, 60, 90, 120 and 150 min. A cutoꢀ oꢁ
1
8 s was set to prevent the risk oꢁ burns. The results were
expressed as the mean± SEM in seconds at diꢀerent times.
Hot plate test
The hot plate test was perꢁormed as described previously by
Woolꢁe and MacDonald (1944). This test is a model ꢁor stud-
ying supra-spinal-acting analgesic compounds. The latency
6
number oꢁ leukocytes × 10 /ml. In addition, another ali-
quot was used to determine the activity oꢁ myeloperoxidase
enzyme and to quantiꢁy the TNF-α and IL-1β levels. How-
ever, only the experimental groups that presented a reduction
in the total or diꢀerential number oꢁ leukocytes were evalu-
ated in the test oꢁ myeloperoxidase enzyme activity and in
the assay ꢁor quantiꢂcation oꢁ cytokines.
(
in seconds) in reaction to the thermal stimulus, expressed
by liꢁting oꢁ the hind paws accompanied by licking or finch-
®
ing, was measured using a hot plate (Insight , Ribeirão
Preto, São Paulo, Brazil) set at 55.5 ± 0.5 °C. The mice
were divided into ꢁour experimental groups (n=8): vehicle
(
10% DMSO, 10 mL/kg, p.o.), LQFM-102 (3) (0.6 mmol/
kg, p.o.), paracetamol (2) (0.6 mmol/kg, p.o.) or morphine
0.017 mmol/kg, s.c.—positive control ꢁor antinociceptive
Activity of myeloperoxidase enzyme
(
activity). The latency baseline was perꢁormed 30 min beꢁore
the treatments (time − 30) and immediately aꢁter the treat-
ments (time 0) ꢁor each animal. Subsequently, the latency to
pain reaction was measured at 30, 60, 90, 120 and 150 min.
A cutoꢀ oꢁ 20 s was set to prevent the risk oꢁ burns. The
results were expressed as the mean ± SEM in seconds at
various times.
To measure the myeloperoxidase (MPO) activity, 40 µL oꢁ
pleural lavage oꢁ mice treated with vehicle (10% DMSO,
10 mL/kg, p.o.), LQFM-102 (3) (0.6 mmol/kg, p.o.) or
celecoxib (1) (0.6 mmol/kg, p.o.) was transꢁerred to Eppen-
dorꢁ tubes. The reaction was started by adding 360 µL
oꢁ phosphate buꢀer, pH 6.0, containing 0.167 mg/mL oꢁ
o-dianisidine 2HCl and 0.0005% H O . The enzyme reac-
2
2
tion was stopped aꢁter 15 min by adding 30 µL oꢁ 1% sodium
azide. The samples were subsequently centriꢁuged ꢁor 5 min
at 1000 rpm. The supernatant was separated, and 200 µL was
transꢁerred to microplate wells, and absorbance was assayed
by enzyme-linked immunosorbent assay (ELISA) reading at
a wavelength oꢁ 450 nm (Saleh, et al. 1999; Sedgwick 1995).
The results are expressed as the mean± SEM oꢁ enzymatic
activity in mU/mL.
Carrageenan‑induced oedema
Carrageenan-induced hind paw oedema in mice was used
as the animal model oꢁ acute infammation according to
the method oꢁ Winter et al. (1962). Initially, the animals
(
n=8) were treated with vehicle (10% DMSO, 10 mL/kg,
p.o.), LQFM-102 (3) (0.6 mmol/kg, p.o.), paracetamol (2)
0.6 mmol/kg, p.o.) or celecoxib (1) (0.6 mmol/kg, p.o.). 1
(
h later, 50 µL oꢁ carrageenan (1%) was injected intraplantar
to the right hind paw and 50 µL oꢁ saline (0.9% NaCl) was
injected intraplantar to the leꢁt hind paw (used as the con-
trol). The paw volume was measured using a plethysmom-
eter (Model 7141, Ugo Basile, Italy) at 1, 2, 3, and 4 h aꢁter
carrageenan injection. The baseline was determined beꢁore
the treatments (time 0) ꢁor each animal. The results were
expressed in µL as the mean± SEM.
Assay of TNF‑α and IL‑1β levels
The pleural exudates oꢁ mice treated with vehicle (10%
DMSO, 10 mL/kg, p.o.), LQFM-102 (3) (0.6 mmol/kg,
p.o.) or celecoxib (1) (0.6 mmol/kg, p.o.) were also used
to determine the TNF-α and IL-1β concentrations using an
immunosorbent assay kit (ELISA) (Ebioscience) (Nicoletti
et al. 2010; Costa et al. 2013). The results were expressed as
the mean ± SEM in pg/mL.
Carrageenan‑induced pleurisy
Pharmacological evaluation from treatment
with repeated doses
The animals (n=8) were treated with vehicle (10% DMSO,
1
0 mL/kg, p.o.), LQFM-102 (3) (0.6 mmol/kg, p.o.), par-
acetamol (2) (0.6 mmol/kg, p.o) or celecoxib (1) (0.6 mmol/
kg, p.o.). 1 h aꢁter the treatments, the animals received an
injection oꢁ 100 μL oꢁ 1% carrageenan into the pleural cav-
ity. 4 h later, the pleural exudate was collected with 1 mL
oꢁ heparinized phosphate-buꢀered saline (Vinegar et al.
Experimental protocol
To evaluate the eꢀect oꢁ chronic treatment with LQFM-102
(3) on the infammatory process and some parameters oꢁ tox-
icity, the animals (n=8) were treated once daily ꢁor 15 days
with vehicle (10% DMSO, 10 mL/kg, p.o.), LQFM-102 (3),
1973). One aliquot was used to determine the total leukocyte
1
3

D. P. B. da Silva et al.
paracetamol (2) or celecoxib (1) at 0.3 mmol/kg, p.o. At the
penultimate day oꢁ treatment, the animals were ꢁasted. At
the last day oꢁ treatment, the animals were submitted to the
ear oedema induced by croton oil test (60 min aꢁter the treat-
ments), and were euthanized by decapitation, completing the
minute (with 15-s breaks) using IKA-ULTRATURRAX T8
homogenizer. For determination oꢁ GSH level, 950 μL oꢁ the
studied homogenate was ꢂrst deproteinized by the addition
oꢁ 50 μL oꢁ cold 50% trichloroacetic acid (TCA) and then
the sample was centriꢁuged at 10 000×g at a temperature
oꢁ + 4 °C ꢁor 10 min. To 850 μL oꢁ 0.2 M phosphate buꢀer
(pH 8.2), 100 μL oꢁ 6 mM 5.5′-dithiobis(2-nitrobenzoic
acid) (DTNB) and 50 μL oꢁ supernatant ꢁrom deprotein-
ized homogenate were added. Absorbance was measured at
a wavelength λ=412 nm, 1 min aꢁter supernatant addition.
The total contents oꢁ glutathione (GSH) were determined
ꢁast 12 h beꢁore euthanasia. Immediately aꢁter euthanasia, we
collected plasma oꢁ animals ꢁor evaluation oꢁ serum AST,
ALT, urea and creatinine; ears ꢁor oedema measurement;
liver ꢁor quantiꢂcation oꢁ glutathione and stomach ꢁor inves-
tigation oꢁ gastric toxicity.
ꢁ
rom a standard curve prepared ꢁor the 1 mM GSH and were
expressed as the mean ± SEM in μmol per gram oꢁ tissue
Kowalczyk-Pachel et al. 2016).
Measurement of body weight
(
During the procedure, the animals’ weights were monitored
daily during the 15-day experimental procedure and a sig-
niꢂcant change in corporal weight was considered as an indi-
cator oꢁ a toxic or side eꢀect ꢁor LQFM-102 and dipyrone
treatments (Yu et al. 2006).
Gastric lesion assessment
The stomach was collected and opened along the small cur-
vature to assess the lesion index. The assessment oꢁ lesion
index was conducted in a blinded manner and calculated by
adding the ꢁollowing values obtained in the observation oꢁ
mucosa, considering the lesion degree produced: light (1
point), moderate (2 points) or intense (3 points), evaluat-
ing the discoloration oꢁ mucosa, the presence oꢁ petechial,
hemorrhages, mucus loss and number oꢁ ulcers (Martins
et al. 2014).
Ear oedema induced by croton oil
Ear oedema was induced by croton oil based on previous
studies (Romay et al., 1998 and Tubaro et al., 1986) with
some modiꢂcations. 1 h aꢁter the last treatment with vehicle
(
(
10% DMSO, 10 mL/kg, p.o.), LQFM-102 (3), paracetamol
2) or celecoxib (1) at 0.3 mmol/kg, p.o., 2.5% croton oil in
acetone (20 μL/ear) was administered by topical application
in the right ear oꢁ each mouse. The same volume (20 μL) oꢁ
acetone was applied to the leꢁt ear. 4 h aꢁter the application
oꢁ the phlogistic agent, mice were euthanized by decapita-
tion and both ears were cut at the base and the weight diꢁ-
In vitro assay to determine IC of LQFM‑102 (3)
50
for COX and 5‑LOX
The COX-1, COX-2 and 5-LOX inhibition assays were
perꢁormed according to the protocol instructions oꢁ the
Colorimetric COX (Ovine) Inhibitor Screening Assay Kit
and the Lipoxygenase (soybean) Inhibitor Screening Assay
Kit (Cayman Chemical Company, MI, USA, Catalog Nos.
ꢁ
erence between the samples oꢁ the control ear (leꢁt) and the
croton oil-treated ear (right) was established on an analytical
balance (Swingle et al. 1981). The results were expressed as
the mean ± SEM oꢁ variation oꢁ the oedema, in percentage.
7
60111 and 760700, respectively).
(
Weight of the right air − Weight of the left ear)
Δ =
× 100.
Weight of the left ear
Statistical analysis of pharmacological tests
Hepatic and renal function analysis
The data were analyzed statistically by one-way ANOVA
ꢁ
ollowed by the Newman–Keuls’ post hoc test or two-way
Serum alanine aminotransꢁerase (ALT) and aspartate ami-
notransꢁerase (AST) activities as well as serum urea and
creatinine levels were measured as indicators oꢁ hepatic
and renal injury, respectively. These parameters were meas-
ured using standard enzymatic methods (Doles reagent kits,
Brazil).
ANOVA ꢁollowed by the Bonꢁerroni post hoc test (Sokal and
Rohlꢁ 1981). All statistical analyses were carried out using
the GraphPad Prism version 5.0. Values oꢁ P≤ 0.05 were
considered signiꢂcant.
Structural analysis
Determination of GSH in the liver
The crystal structure oꢁ human COX-2 (PDB ID: 5IKV)
(
Orlando and Malkowski 2016) was obtained ꢁrom the Pro-
The liver tissue samples were chilled at 4 °C, weighted and
homogenized (1 g oꢁ the tissue in 4 mL oꢁ 0.1 M phos-
phate buꢀer, pH 7.4) at a speed oꢁ 25 000 revolutions per
tein Data Bank (Berman et al. 2000). Since there was no
structure oꢁ the human COX-1 available, we built a model
based on ovine COX-1 (PDB ID: 3N8X) (Sidhu et al. 2010),
1
3

Molecular docking and pharmacological/toxicological assessment of a new compound designed…
which was identical to other models deposited to the Pro-
tein Model Portal (Haas et al. 2013). The heme groups
were added ꢁor both COX-1 and COX-2. To limit the search
to 1-(4-nitrophenyl)-1H-pyrazole-4-carboxylic acid (7)
with a 74% yield (Kleiderer and Shriner 1943). At the next
step, 1-(4-nitrophenyl)-1H-pyrazole-4-carboxylic acid (7)
was esteriꢂcated through Fischer’s conditions to methyl
1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate (8) with a
94% yield (Furniss et al. 1996). The nitro-group present in
methyl 1-(4-nitrophenyl)-1H-pyrazole-4-carboxylate (8) was
ꢁ
or ligand poses to the vicinity oꢁ active sites, we used a
grid centered on the ligands on 3N8X and 5IKV. Redock-
ing was perꢁormed using both DockThor (Custódio et al.
2
2
3
014; de Magalhães et al. 2014) and Vina (Trott and Olson
010), with three structures ꢁor COX-1 (1Q4G, 2AYL and
KK6) and COX-2 (3NT1, 4PH9 and 5JW1). LQFM-102
reduced with NaBH in MeOH using 10% palladium-on-
4
carbon as a catalyst to obtain methyl 1-(4-aminophenyl)-
1H-pyrazole-4-carboxylate (9) with a 45% yield (Petrini
et al. 1987). The acetylation oꢁ amino group ꢁrom methyl
1-(4-aminophenyl)-1H-pyrazole-4-carboxylate (9) was car-
ried out with acetic anhydride and iodine as a catalyst with
a 44% yield (Borah et al. 1997). At the end, the hydrolysis oꢁ
ester moiety ꢁrom methyl 1-(4-acetamidophenyl)-1H-pyra-
zole-4-carboxylate (10) to 1-(4-acetamidophenyl)-1H-pyra-
zole-4-carboxylic acid (3) was carried out with KOH in
was drawn in Avogadro, with its partial charges calculated
by Gaussian (Frisch et al. 2009) using MP2, serving as an
input ꢁor molecular docking with DockThor and Vina. The
same methodology was employed to dock paracetamol and
celecoxib to both COX-1 and COX-2, allowing the com-
parison oꢁ binding poses and contacts oꢁ each molecule with
the proteins. Interactions between protein and ligands were
evaluated with PoseView [58] and images were created with
PyMol (The PyMOL Molecular Graphics System, version
THF:MeOH:H O medium at room temperature, with a 67%
2
1
.8).
yield (Khurana et al. 2004). LQFM-102 (3) was obtained in
seven synthetic steps with an 8.9% overall yield.
Results
Pharmacological evaluation
Synthesis of LQFM‑102 (3)
Formalin test
As illustrated in Fig. 1b, the synthetic route began
with 1-(4-nitrophenyl)-1H-pyrazole (5) and proceeded
through the classical method described by Finar and
Godꢁrey and resulted in an 88% yield (Finar and Godꢁrey
The oral treatments with LQFM-102 (3) showed a signiꢂcant
antinociceptive eꢀect when compared to the control group
in the ꢂrst test phase (ANOVA: F
=9.618, P<0.0001)
(
5,47)
and the second test phase (ANOVA: F
= 9.897,
5,47)
(
1
1
1
954). Chemoselective and regiospeciꢂc ꢁormylation oꢁ
-(4-nitrophenyl)-1H-pyrazole (5) to 1-(4-nitrophenyl)-
Hpyrazole-4-carbaldehyde (6) was perꢁormed under
P < 0.0001) (Fig. 2). In the ꢂrst phase oꢁ ꢁormalin test,
animals treated with vehicle (mL/kg p.o.) showed a lick-
ing time (s) oꢁ 81.2 ± 4.1, and LQFM-102 (3) at doses oꢁ
0.3, 0.6 or 1.2 mmol/kg, reduced this time to 51.7 ± 6.0
(36.3% less), 46.8± 5.0 (42.4% less) and 43.3± 7.1 (46.6%
less), respectively. In the second phase, the vehicle group
Duꢀ’s conditions with an 83% yield (Oliveira et al. 2013).
In turn, 1-(4-nitrophenyl)-1H-pyrazole-4-carbaldehyde (6)
was oxidated using KMnO in water at a refux temperature
4
Fig. 2 Antinociceptive eꢀect oꢁ LQFM-102 (3), celecoxib (1) or paracetamol (2) in the ꢂrst phase and second phase oꢁ ꢁormalin test. *P≤0.05,
*P≤0.01 and ***P≤0.001 compared to control group, according to ANOVA ꢁollowed by post hoc Newman–Keuls’ test
*
1
3

D. P. B. da Silva et al.
showed a licking time (s) oꢁ 207.3± 11.1, and LQFM-102
(
(
3) reduced this time to 146.3± 18.2 (29.4% less), 135± 6.1
34.9% less) and 83.5± 8.9 (59.7% less), respectively. The
positive control group treated with paracetamol (0.6 mmol/
kg, p.o.) decreased the ꢂrst phase to 52.3± 3.0 (35.6% less)
and the second phase to 161± 17.1 (a reduction oꢁ 22.3%).
The anti-infammatory positive control group treated with
celecoxib (1) (0.6 mmol/kg, p.o.), as expected, decreased the
licking time (s) only in the second phase, to 148.4± 10.3 (a
reduction oꢁ 28.4%).
Tail ꢀick test
In the thermal nociception tail fick test, treatment with
LQFM-102 (3) (0.6 mmol/kg, p.o.) or paracetamol (2)
(
0.6 mmol/kg, p.o.) maniꢁested no signiꢂcant antinociceptive
activity when compared to the control group (vehicle). The
opioid agonist morphine (0.017 mmol/kg, s.c.) demonstrated
signiꢂcant antinociception at the time oꢁ 30, 60, 90, 120 and
1
50 min aꢁter treatment (Fig. 3a).
Hot plate test
In the thermal nociception hot plate test, treatment with
LQFM-102 (3) (0.6 mmol/kg, p.o.) or paracetamol (2)
(
0.6 mmol/kg, p.o.) maniꢁested no signiꢂcant antinocicep-
tive activity when compared to the control group (vehicle).
The morphine (0.017 mmol/kg, s.c.—opioid agonist) dem-
onstrated signiꢂcant antinociception at times oꢁ 30, 60, 90,
1
20 and 150 min aꢁter treatment (Fig. 3b).
Carrageenan‑induced oedema
Treatment with LQFM-102 (3) reduced the carrageenan-
induced paw oedema at all time points oꢁ the test (ANOVA:
F
= 53.43, P < 0.0001). In the ꢁirst hour, treatment
3140)
(
Fig. 3 Eꢀect oꢁ LQFM-102 (3), paracetamol (2) or morphine on
a tail fick and b hot plate test. ***P≤0.001, compared to control
group, compared to control group, according Two-way ANOVA ꢁol-
lowed by Bonꢁerroni’s test
with LQFM-102 (3) (0.6 mmol/kg, p.o.) or celecoxib (1)
(0.6 mmol/kg, p.o.) reduced the oedema to 107± 3.3 (21.2%
reduction) or 112.2± 3.6 (17.4% reduction), respectively,
compared to the control group (diꢁꢁerence between the
paws oꢁ 135.8 ± 3.6 µl). These same treatments reduced
the oedema to 94± 5.2 (27.7% reduction) or 104.6± 3.3 µl
Cell migration
(
19.5% reduction) in the second hour, 80± 5.8 (35.1% reduc-
In the test oꢁ carrageenan-induced pleurisy, the treat-
ment with LQFM-102 (3) (0.6 mmol/kg) or celecoxib (1)
(0.6 mmol/kg) reduced the total number oꢁ leukocytes to
5.365± 0.52 (32.3% reduction) or 4.330± 0.69 leukocytes
tion) or 85.4 ± 5.2 µl (30.7% reduction) in the third hour,
and 77 ± 6.8 (32.7% reduction) or 73.7 ± 6.8 µl (reducing
by 35.6%) in the ꢁourth hour oꢁ the test compared to the
control group (diꢀerence between the paws oꢁ 130± 4.43,
6
× 10 /mL (reduction by 45.3%), respectively, compared
6
1
23.3 ± 3.7, and 114.4 ± 5.7 µl, respectively). The group
to the control group (7.920 ± 0.38 leukocytes x 10 /mL)
treated with paracetamol (2) (0.6 mmol/kg, p.o.) did not
(ANOVA: F
=31.6, P<0.0001). According to the diꢁ-
4,39)
(
reduce the carrageenan-induced paw oedema (Fig. 4a).
ꢁerential count, these reductions were due to a decrease in
1
3

Molecular docking and pharmacological/toxicological assessment of a new compound designed…
Fig. 4 Eꢀect oꢁ LQFM-102 (3), paracetamol (2) or celecoxib (1) on
a paw oedema induced by carrageenan; b total number oꢁ leukocytes;
c number polymorphonuclears cells migrated and d myeloperoxidase
activity when compared with the control group. *P≤0.05, **P≤0.01
and ***P≤0.001, compared to control group, compared to control
group, according One-way ANOVA ꢁollowed by Newman–Keuls’s
test or Two-way ANOVA ꢁollowed by Bonꢁerroni’s test
polymorphonuclear cells (ANOVA: F(2,23) =8.6, P<0.0001)
Assay of TNF‑α and IL‑1β levels
to 4.190± 0.38 (34% reduction) or 5.042± 0.35 leukocytes
6
×
10 /mL (reduction by 21%), respectively, compared to the
The level oꢁ TNF-α was reduced by treatment with
6
control group (6.361± 0.39 leukocytes × 10 /mL) (Fig. 4c).
LQFM-102 (3) (0.6 mmol/kg) (ANOVA: F
= 15.97,
3,23)
(
As shown the Fig. 4b, c, treatment with paracetamol (2)
P < 0.0001) to 16.6 ± 3.3 (37.6% reduction), compared
to the vehicle-treated group (TNF-α: 26.6 ± 3.6 pg/mL).
However, the treatment with celecoxib (1) (0.6 mmol/
kg) did not reduce the levels oꢁ TNF-α in pleural exudate
as seen in Fig. 5a. As can be observed in Fig. 5b, these
same treatments signiꢂcantly reduced the levels oꢁ IL-1β
(
0.6 mmol/kg, p.o.) did not reduce cell migration.
Activity of myeloperoxidase enzyme
Treatment with LQFM-102 (3) (0.6 mmol/kg) or celecoxib
(
1) (0.6 mmol/kg) also reduced the activity oꢁ myelop-
(ANOVA: F
= 381.8, P < 0.0001) to 481.4 ± 19.2
3,23)
(
eroxidase enzyme to 73.2 ± 5.7 (39.2% reduction) or
0.8 ± 4.5 mU/mL (57.8% reduction), respectively, com-
pared to the vehicle-treated group (enzymatic activity oꢁ
20.5± 14.5 mU/mL) (ANOVA: F( =31.97, P<0.0001)
(16.8% reduction) or 467.3± 5.5 pg/mL (19.3% reduction),
respectively, compared to the vehicle-treated group (IL-1β:
579 ± 13.3 pg/mL).
5
1
3,31)
(
Fig. 4d).
1
3

D. P. B. da Silva et al.
A
40
30
20
*
10
0
Vehicle
0 mL/kg
Vehicle
10 mL/kg
LQFM-102
0.6 mmol/kg
Celecoxib
0.6 mmol/kg
1
_
____ ___________________
Saline 0.9%
Carrageenan 1%
B
8
00
00
00
6
***
***
4
200
0
Vehicle
0 mL/kg
Vehicle
10 mL/kg
LQFM-102
0.6 mmol/kg
Celecoxib
0.6 mmol/kg
1
_
____ ___________________
Saline 0.9%
Carrageenan 1%
Fig. 5 Eꢀect oꢁ LQFM-102 (3) or celecoxib (1) on a TNF-α lev-
els and b IL-1β levels, in the pleural exudate. *P<0.05 and
*
**P<0.001, compared to control group, according ANOVA ꢁol-
lowed by Newman–Keuls’s test
Fig. 6 Eꢀect oꢁ chronic treatment with LQFM-102 (3), celecoxib
(
1) or paracetamol (2) on a body weight oꢁ animals, and b the croton
oil-induced ear oedema. ***P≤0.001, compared to control group,
according One-way ANOVA ꢁollowed by Newman–Keuls’s test or
Two-way ANOVA ꢁollowed by Bonꢁerroni’s test
Pharmacological evaluation from treatment
with repeated doses
Measurement of body weight
Ear oedema induced by croton oil
The body weight was evaluated during 15 days oꢁ the
treatments with either vehicle (10 mL/kg), LQFM-102
The chronic treatment with LQFM-102 (3) showed a sig-
niꢂcant anti-infammatory activity maniꢁested in a reduc-
tion oꢁ ear oedema compared to the control group (ANOVA:
(
3) (0.3 mmol/kg), paracetamol (2) (0.3 mmol/kg) or
celecoxib (1) (0.3 mmol/kg). No changes in total body
F
=24.43, P<0.0001). The daily treatment with LQFM-
3,31)
(
weight were observed compared with the vehicle group
102 (3) (0.3 mmol/kg), paracetamol (2) (0.3 mmol/kg)
(
Fig. 6a).
or celecoxib (1) (0.3 mmol/kg) ꢁor 15 days signiꢂcantly
1
3

Molecular docking and pharmacological/toxicological assessment of a new compound designed…
reduced the percentage oꢁ the weight diꢀerence between the
animal ears to 40.5± 3 (66.6% reduction), 73.2± 3.6 (39.6%
reduction), or 56.5 ± 6.7 (53.4% reduction), respectively,
compared to the control group (Δ oedema: 121.3± 11.5%)
(
Fig. 6b).
Hepatic and renal function analysis
The AST, ALT, urea and creatinine levels were assayed in
blood samples ꢁrom animals subchronically treated with
vehicle (10 mL/kg, p.o.), LQFM-102 (3) (0.3 mmol/kg,
p.o.), paracetamol (2) (0.3 mmol/kg, p.o.) or celecoxib (1)
(
ꢁ
0.3 mmol/kg, p.o.) ꢁor 15 days. Serum levels oꢁ the hepatic
unction indicators AST (ANOVA: F(
=8.26, P<0.0009)
3,23)
and ALT (ANOVA: F
=8.886, P<0.0006) and levels oꢁ
3,23)
(
the renal ꢁunction indicators urea (ANOVA: F(
=7.216,
3,23)
= 5.608,
3,23)
P < 0.0018) and creatinine (ANOVA: F
(
P<0.0059) were not modiꢂed by treatment with LQFM-102
(
3). However, the treatment with paracetamol (2) increased
the level oꢁ all indicators (AST: 145.5% increase; ALT:
8.9% increase; urea: 24.3% increase and creatinine: 114.8%
5
increase) compared with the control group. Treatment with
celecoxib (1) signiꢂcantly increased only the levels oꢁ urea
and creatinine (urea: 18.9% increase and creatinine: 138.5%
increase) compared with the control group (Table 1).
Determination of GSH in the liver
The chronic treatment with LQFM-102 (3) (0.3 mmol/kg,
p.o.) or celecoxib (1) (0.3 mmol/kg, p.o.) did not show sig-
niꢂcant alteration in the GSH levels in animal livers when
compared to the control group (GSH: 11.2 ± 0.8 µmol/g
oꢁ tissue). The chronic treatment with paracetamol (2)
(
0.3 mmol/kg, p.o.) demonstrated a signiꢂcant decrease
in the GSH levels to 8.1 ± 0.6 µmol/g oꢁ tissue (27.7%
reduction) compared to the control group (ANOVA:
Fig. 7 Eꢀect oꢁ chronic treatment with LQFM-102 (3), celecoxib (1)
or paracetamol (2) on a GSH levels in the liver and b index oꢁ gas-
tric lesions. * P≤0.05 and ** P≤0.01, compared to control group,
according One-way ANOVA ꢁollowed by Newman–Keuls’s test
F
=3.816, P<0.0175) (Fig. 7A).
3,31)
(
Table 1 Eꢀect oꢁ chronic treatment with LQFM-102 (3), celecoxib (1) or
paracetamol (2) at dose 0.3 mmol/kg once daily ꢁor 15 days on plasma markers AST, ALT, urea and creatinine
Plasma biomarkers indicative oꢁ toxicity (Mean± SEM)
Treatment once daily ꢁor 15 days
Vehicle 10 mL/kg
LQFM-102
Paracetamol 0.3 mmol/kg
Celecoxib 0.3 mmol/kg
0
.3 mmol/kg
AST (U.I./L)
ALT (U.I./L)
Urea (mmol/L)
Creatinine (µmol/L)
30.1± 3.4
31.4± 3.2
7.4± 0.4
38.4± 3.9
29.2± 3.2
7.5± 0.3
73.9± 11.6**
49.9± 4.0**
9.2± 0.3**
38.4± 5.9
32.6± 2.1
8.8± 0.4*
498.3± 80*
208.9± 64
194.2± 38
448.7± 77.2*
*
p ≤ 0.05; **p ≤ 0.01
1
3

D. P. B. da Silva et al.
Gastric lesion assessment
residues (His90, Ser353, Phe518, Val523 and Ala527) and
Leu352, important ꢁor potency (Fig. 9a, c, ꢁ). With Vina,
LQFM-102 (3) interacted with the catalytic residues Val349
and Tyr385, the stabilizing residues Arg120 and Tyr355, and
with Ala527 and Leu531 (Fig. 9b, c, i). Overall, LQFM-
102 (3) ꢁared better than any other ligand tested, interacting
with more key residues than paracetamol (2) or celecoxib
(1) (Figs. 8d–i, 9d–i).
Administration oꢁ LQFM-102 (3) (0.3 mmol/kg) or
celecoxib (1) (0.3 mmol/kg) daily ꢁor 15 days did not induce
signiꢂcant gastric lesions, as shown in Fig. 7b. On the other
hand, paracetamol (2) (0.3 mmol/kg daily ꢁor 15 days)
resulted in damage to the stomach mucosa with an increase
in the index oꢁ gastric lesions to 13.7± 1.5 (64.5% increase)
compared to the control group (8.3 ± 0.8 index oꢁ gastric
lesions), (ANOVA: F(
=4.331, P<0.0140).
3,31)
In vitro assay to determine IC of LQFM‑102 (3)
Discussion
5
0
for COX and 5‑LOX
The ꢁormalin-induced pain test was conducted to evaluate
and diꢀerentiate antinociceptive eꢀect oꢁ LQFM-102 (3).
As described in the literature, both phases oꢁ this test can
be inhibited by central or peripheral analgesics, while anti-
infammatory drugs inhibit only the second phase oꢁ the test
(Hunskaar and Hole 1987; Le Bars et al. 2001). LQFM-102
(3) signiꢂcantly reduced the licking time in both phases oꢁ
the test, suggesting that this compound retained a satis-
ꢁactory analgesic and anti-infammatory activity ꢁrom the
precursors. In the next tests, only the intermediate dose oꢁ
LQFM-102 (3) was employed, with the aim to reduce the
number oꢁ animals as recommended by the CEUA.
LQFM-102 (3) inhibited both COX-1 and COX-2, with the
COX-2 selectivity index similar to that oꢁ celecoxib (1) and
higher than that oꢁ paracetamol (2), according to the IC
5
0
values described in the literature ꢁor both precursors. In
addition, LQFM-102 (3) also inhibited the 5-Lox enzyme,
as seen in Table 2.
Structural analysis
Redocking with COX-1 and COX-2 perꢁormed with the
docking programs DockThor and Vina repositioned the
cocrystalized ligands close to their original positions. Fol-
lowing the same methodology, both DockThor and Vina
positioned LQFM-102 (3) in the active site oꢁ each pro-
tein. Docking positioned LQFM-102 (3) in such a way
that it interacted with the key residues oꢁ both COX-1 and
COX-2 (Sidhu et al. 2010; Orlando and Malkowski 2016;
Kothekar et al. 2001). Regarding COX-1 (Fig. 8), docking
with DockThor resulted in LQFM-102 (3) interactions with
the catalytic residue Ser530, the ligand-stabilizing residue
Arg120, as well as with the cavity-ꢁorming residues Ile523
and Ala527 (Fig. 8a, c, ꢁ). Docking with Vina showed that
LQFM-102 (3) also interacted with the catalytic residues
Val349 and Ser530, the cavity-ꢁorming residues Met522 and
Ala527, and with Leu352, shown to be important ꢁor NSAID
potency (Fig. 8b, c, i). For COX-2 (Fig. 9), DockThor placed
paracetamol (2) apart ꢁrom other ligands in such a way that it
only interacted with Glu524 (Fig. 9a, d). For LQFM-102 (3),
predicted contacts included Val349, several cavity-ꢁorming
To investigate possible involvement oꢁ the central mech-
anism in antinociceptive eꢀect oꢁ LQFM-102 (3) seen in
the ꢂrst phase oꢁ ꢁormalin test, the tail fick and hot plate
tests were conducted. Both tests used a thermal stimulus
to evaluate antinociceptive activity mediated by a spinal
and/or supra-spinal mechanism, respectively (Barrot 2012;
D’amour and Smith 1941; Woolꢁe and MacDonald 1944).
Paracetamol (2) at the dose used did not increase the thermal
latency in both tests. However, some studies have reported
that at higher doses; paracetamol produces eꢀect in these
tests, mainly due to activation oꢁ descending serotonergic
pathways (Dogrul et al. 2012; Pini et al. 1996; Sandrini
et al. 2003). Furthermore, previous studies have shown an
involvement oꢁ peripheral mechanisms in the analgesic eꢀect
oꢁ paracetamol (2), which blocks the generation oꢁ action
potentials in the nociceptive peripheral ꢂbers (Anderson
2008; Dani et al. 2007). In both tests, LQFM-102 (3) also
did not increase the latency in response to thermal stimuli,
Table 2 The IC oꢁ LQFM-
5
0
LQFM-102
IC50 (µM)
1
5
02 (3) ꢁor COX-1, COX-2 and
-LOX enzymes
COX-1
982.0
COX-2
5.8
5-LOX
91.6
1
Selectivity index–LQFM-102 (COX-1/COX-2)
Selectivity index–Celecoxib (COX-1/COX-2)
Selectivity index–Paracetamol (COX-1/COX-2)
341.7
313.3
0.023
Al-Hourani et al. (2011)
Chandrasekharan et al. (2002)
1
3

Molecular docking and pharmacological/toxicological assessment of a new compound designed…
Fig. 8 Docking and contacts oꢁ COX-1 with paracetamol (2),
celecoxib (1) and LQFM-102 (3). Best poses oꢁ paracetamol (red/pur-
ple), celecoxib (green) and LQFM-102 (blue) with DockThor (a) and
Vina (b), with LQFM-102 on detail (c). Interactions are shown ꢁor
DockThor (d–f) and Vina (g–i) ꢁor paracetamol (d, g), celecoxib (e,
h) an LQFM-102 (f, i)
suggesting that the antinociceptive eꢀect oꢁ LQFM-102 (3)
at the dose used was due to peripheral mechanisms.
The paw oedema induced by carrageenan test was con-
ducted to evaluate the anti-infammatory eꢀect oꢁ LQFM-
which limits the ability oꢁ the drug to inhibit the cyclooxy-
genase enzymes (Graham et al. 2013; Whitehouse and But-
ters 2014). On the other hand, LQFM-102 (3) promoted the
reduction oꢁ oedema independently oꢁ the peroxide presence
at the infammation site.
1
02 (3). Treatment with LQFM-102 (3) signiꢂcantly reduced
the oedema ꢁormation, similarly to celecoxib (1). However,
paracetamol (2) at the dose used had no eꢀect on the oedema
A series oꢁ new pyrazole derivatives have been devel-
oped and that have demonstrated excellent anti-infamma-
tory activities (Khan et al. 2016), speciꢂcally, inhibition
oꢁ paw oedema ꢁormation at all time points oꢁ the test
(Chavan et al. 2013; Oliveira et al. 2016) and reducing
ꢁ
ormation. This was because paracetamol (2) exhibits low
anti-infammatory activity due to the presence oꢁ high per-
oxide concentrations produced at the site oꢁ infammation,
1
3

D. P. B. da Silva et al.
Fig. 9 Docking and contacts oꢁ COX-2 with paracetamol (2),
celecoxib (1) and LQFM-102 (3). Best poses oꢁ paracetamol (red/pur-
ple), celecoxib (green) and LQFM-102 (blue) with DockThor (a) and
Vina (b), with LQFM-102 on detail (c). Interactions are shown ꢁor
DockThor (d–f) and Vina (g–i) ꢁor paracetamol (d, g), celecoxib (e,
h) an LQFM-102 (f, i)
some pro-inꢁlammatory parameters in carrageenan-
induced pleurisy test (Florentino et al. 2017).
(Desmarchelier et al. 1997; Eddouks et al. 2012), conꢂrm-
ing the result oꢁ total/diꢀerential count.
The pleurisy test allows the evaluation and quantiꢂ-
cation oꢁ infammatory parameters, such as cell migra-
tion, myeloperoxidase activity and cytokine levels in the
collected pleural exudate (Higgs et al. 1980; Mikami and
Miyasaka 1983; Vinegar et al. 1973). It was observed in
this test that LQFM-102 (3) or celecoxib (1) reduced cell
migration, mainly, by reducing polymorphonuclears cell
migration. Paracetamol (2) at an equimolar dose did not
signiꢂcantly reduce these parameters. Moreover, LQFM-
In this sense, it is possible to suggest that the anti-
infammatory eꢀect oꢁ LQFM-102 (3) involves a reduc-
tion and/or blockage oꢁ the release oꢁ infammatory media-
tors that promote leucocyte chemotaxis, such as cytokines
(Frode et al. 2001; Mazzon and Cuzzocrea 2007) and
chemokines (Kobayashi, 2008; Sanz and Kubes, 2012).
Thereꢁore, the next step was to investigate the eꢀect oꢁ
LQFM-102 (3) on TNF-α and IL-β levels in pleural exu-
dates and, as expected, the new pyrazole compound sig-
niꢂcantly reduced the levels oꢁ both cytokines, in contrast
with celecoxib that reduced only the IL-β levels. Similar
results in relation to celecoxib (1) were obtained in the
same experimental model by Pinheiro and Calixto (2002),
1
02 (3) or celecoxib (1) reduced the activity oꢁ the mye-
loperoxidase enzyme, which is predominantly present
in active neutrophils and its levels are directly propor-
tional to the neutrophil concentration in infamed tissue
1
3

Molecular docking and pharmacological/toxicological assessment of a new compound designed…
which showed that this compound had a low inhibitory
eꢀect on the production oꢁ TNF-α and IL-β at the infam-
mation site. Furthermore, other studies have shown a non-
signiꢂcant eꢀect oꢁ celecoxib (1) on the TNF-α level in
diꢀerent doses and experimental protocols (De Souza et al.
Computational analysis by molecular docking also reveals
the ability oꢁ LQFM-102 (3) to interact with the key residues
ꢁor activity and potency, perꢁorming better than paracetamol
(2) and celecoxib (1) in the same simulation conditions. In
all LQFM-102 (3) dockings, residues ꢁrom the active site
were positioned close to the ligand, suggesting interactions
that might inhibit COX activity. Furthermore, interactions
with the key residues that ꢁorm and stabilize the active
cavity might play an important role in ligand positioning.
However, more pharmacological studies are necessary ꢁor
a better understanding oꢁ the mechanisms involved in the
anti-infammatory and antinociceptive eꢀect oꢁ LQFM-
102, as well as ꢁor investigation oꢁ its possible eꢀects on the
cardiovascular system, resulting ꢁrom inhibition oꢁ COX-2
enzyme.
2
015; Niederberger et al. 2001; Page et al. 2016).
Considering that the long-term usage oꢁ NSAIDs may
lead to signiꢂcant side eꢀects (Harirꢁoroosh et al. 2013;
Ungprasert et al. 2012), it was justiꢂed to evaluate the eꢀect
oꢁ chronic treatment with LQFM-102 (3), paracetamol (2)
or celecoxib (1) on the infammatory process, as well as on
the gastric mucosa and markers oꢁ hepatic and renal toxicity.
The results in the ear oedema test showed that all chronic
treatments at equimolar doses maintained the anti-infam-
matory eꢀect; however, LQFM-102 (3) was more eꢀective
than celecoxib (1) and paracetamol (2). In an experiment to
correlate possible hepatic and renal lesions with these treat-
ments, it was observed that only paracetamol increased the
AST and ALT levels. The urea and creatinine levels were
increased by celecoxib and paracetamol. In contrast, LQFM-
Conclusion
In summary, the present ꢂndings suggest that compound
LQFM-102 (3) exerts antinociceptive and anti-infammatory
eꢀects. In ꢁact, this compound reduced pain, hyperalgesia,
oedema, leukocyte migration, myeloperoxidase activity as
well as the TNF-α and IL-1β levels. The antinociceptive and
anti-infammatory eꢀects oꢁ LQFM-102 (3) occurred in the
absence oꢁ alterations in the markers oꢁ hepatic toxicity and
renal toxicity, or lesions oꢁ gastric mucosa. Furthermore, this
compound showed considerable inhibition oꢁ COX-2 and
1
02 (3) caused no changes in these parameters, suggesting
that this new compound is not hepatotoxic or nephrotoxic
at the doses tested.
One oꢁ the most recognized adverse eꢀects oꢁ paraceta-
mol is hepatotoxicity, which may be caused by overdose,
excessive activation oꢁ the cytochrome P450 system, result-
ing ꢁrom the use oꢁ enzyme inducers, or by depletion oꢁ
glutathione (GSH) levels (Junior 2011; Lopes and Matheus
2
012). In this study, it was shown that chronic treatment with
5
-LOX enzymes. Finally, this study shows the therapeutic
LQFM-102 (3) or celecoxib (1) was not able to signiꢂcantly
reduce the GSH levels in the mouse liver, in contrast to the
chronic treatment using paracetamol at the same equimolar
dose, thus corroborating the previous results about hepatic
toxicity indicators, AST and ALT.
potential oꢁ LQFM-102 prototype ꢁor the development oꢁ
analgesic and anti-infammatory drugs.
Acknowledgments The authors are grateꢁul to CNPq (132596/2013-0)
and CAPES (2017OB826336) ꢁor their ꢂnancial support.
Another adverse eꢀect associated with the long-term use
oꢁ NSAIDS is the development oꢁ lesions in gastric mucosa
caused mainly by inhibition oꢁ COX-1 (Henry et al. 1996;
Kodela et al. 2015). The results showed that only treatment
with paracetamol promoted a signiꢂcant increase in the
index oꢁ lesion in the stomach mucosa. The better tolerance
to LQFM-102 (3) in the gastrointestinal tract suggests that
this new compound may have a greater inhibition selectivity
to COX-2 than to COX-1.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conficts oꢁ
interest.
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