Synthesis and antinociceptive evaluation of bioisosteres and hybrids of naproxen, ibuprofen and paracetamol

Article abstract of DOI:10.1016/j.biopha.2018.02.122

The aim of this work was to design, synthesize and characterize the potential anti-nociceptive and anti-inflammatory activities of a new series of bioisosteres and hybrids from known non-steroidal anti-inflammatory drugs (NSAIDs). The compounds 4-(acetylamino)phenyl (2S)-2-(6-methoxy-2-naphthyl)propanoate (GUF-1) and 4-(acetylamino)phenyl 2-(R,S)-(4-isobutylphenyl)propanoate (GUF-2) were synthesized as hybrids (also known as heterodimers); whereas those named 2-(R,S)-(4-isobutylphenyl)-N-1H-tetrazol-5-ylpropanamide (GUF-3), (2S)-2-(6-methoxy-2-naphthyl)-N-1H-tetrazol-5-ylpropanamide (GUF-4), [2-(R,S)-N-hydroxy-2-[4-(2-methylpropyl)phenyl]propanamide] (GUF-5), and (2S)-N-hydroxy-2-(6-methoxy-2-naphthyl)propanamide (GUF-6) were synthesized as bioisosteres of the NSAIDs paracetamol, ibuprofen, and naproxen, respectively. All these compounds were characterized by spectroscopic and spectrometric analysis. Antinociceptive activity of GUF-1 to GUF-6 was evaluated using the formalin test in rats. Pharmacological responses of GUF-1, GUF-2 (hybrids), and GUF-5 (bioisostere) demonstrated significant antinociceptive effects; thus these compounds were assayed in an inflammation test like carrageenan-induced paw oedema in rats. Complete molecular docking of cyclooxygenase and the GUF-1 and GUF-2 hybrids showed high docking scores, compared to the reference drugs. Our data demonstrate that compounds GUF-1, GUF-2, and GUF-5 possesses antinociceptive and antiinflammatory activities resembling and improving those known for the traditional NSAIDs, paracetamol, naproxen and ibuprofen.

Full text of DOI:10.1016/j.biopha.2018.02.122

Biomedicine&Pharmacotherapy101(2018)553–562
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Synthesis and antinociceptive evaluation of bioisosteres and hybrids of
naproxen, ibuprofen and paracetamol
María Eva González-Trujanoa,1, Gerardo Uribe-Figueroab,1, Sergio Hidalgo-Figueroab,
Ana Laura Martíneza, Myrna Déciga-Camposc,⁎, Gabriel Navarrete-Vazquezb,⁎
a
Laboratorio de Neurofarmacología de Productos Naturales. Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñíz”,
Av. México-Xochimilco No. 101, Col. San Lorenzo Huipulco, 14370, Ciudad de México, Mexico
b
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Chamilpa, Cuernavaca, Morelos, 62209, Mexico
Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n, Col. Casco de Santo
c
Tomás, 11340, Ciudad de México, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bioisosteres
Hybrids
The aim of this work was to design, synthesize and characterize the potential anti-nociceptive and anti-in-
flammatory activities of a new series of bioisosteres and hybrids from known non-steroidal anti-inflammatory
drugs (NSAIDs). The compounds 4-(acetylamino)phenyl (2S)-2-(6-methoxy-2-naphthyl)propanoate (GUF-1) and
4-(acetylamino)phenyl 2-(R,S)-(4-isobutylphenyl)propanoate (GUF-2) were synthesized as hybrids (also known
as heterodimers); whereas those named 2-(R,S)-(4-isobutylphenyl)-N-1H-tetrazol-5-ylpropanamide (GUF-3),
(2S)-2-(6-methoxy-2-naphthyl)-N-1H-tetrazol-5-ylpropanamide (GUF-4), [2-(R,S)-N-hydroxy-2-[4-(2-methyl-
propyl)phenyl]propanamide] (GUF-5), and (2S)-N-hydroxy-2-(6-methoxy-2-naphthyl)propanamide (GUF-6)
were synthesized as bioisosteres of the NSAIDs paracetamol, ibuprofen, and naproxen, respectively. All these
compounds were characterized by spectroscopic and spectrometric analysis. Antinociceptive activity of GUF-1 to
GUF-6 was evaluated using the formalin test in rats. Pharmacological responses of GUF-1, GUF-2 (hybrids), and
GUF-5 (bioisostere) demonstrated significant antinociceptive effects; thus these compounds were assayed in an
inflammation test like carrageenan-induced paw oedema in rats. Complete molecular docking of cyclooxygenase
and the GUF-1 and GUF-2 hybrids showed high docking scores, compared to the reference drugs. Our data
demonstrate that compounds GUF-1, GUF-2, and GUF-5 possesses antinociceptive and antiinflammatory ac-
tivities resembling and improving those known for the traditional NSAIDs, paracetamol, naproxen and ibu-
profen.
NSAIDs
Nociception
Inflammation
Formalin test
1. Introduction
simultaneous inhibition of COX-1 largely but not exclusively accounts
for unwanted adverse effects that complicate chronic NSAID therapies.
Non-steroidal anti-inflammatory drugs (NSAIDs) are a chemically
heterogeneous group of compounds sharing certain therapeutic actions
related to nociception, inflammation and pyretic process [1–5]. The
principal therapeutic effect of NSAIDs is the inhibition of cycloox-
ygenase (COX) family proteins that catalyze the rate-limiting steps in
prostaglandin synthesis (PG) [1,2]. The acidic moiety is essential for
COX inhibitory activity and it is linked to a planar aromatic group. The
latter is also connected to a lipophilic part through a polar group. Thus,
NSAIDs are classified as non-selective and COX-2-selective inhibitors
(COXIBs) based on their selectivity for COX inhibition. As a result, the
inhibition of COX-2 is thought to be mediated by the antipyretic, an-
algesic, and anti-inflammatory actions of NSAIDs, while the
The major side effects of NSAIDs include gastrointestinal complications,
renal disturbances and cardiovascular events [6–9].
The market introduction of novel drugs with greater selective in-
hibition on the COX-2 activity in the last decade has offered therapeutic
alternatives with similar efficacy to non-selective inhibitors but better
gastrointestinal tolerability, mainly for patients with chronic use of
traditional NSAIDs. However, concerns regarding the cardiovascular
safety of selective COX-2 inhibitors has led to the withdrawal of two
drugs (rofecoxib and valdecoxib) and focused attention on the non-
specific COX inhibitors. It is now understood that some degree of gas-
trointestinal and cardiovascular risk is associated with the mechanism
of action of all NSAIDs [10–13].
⁎
Corresponding authors.
E-mail addresses: mdeciga@ipn.mx (M. Déciga-Campos), gabriel_navarrete@uaem.mx (G. Navarrete-Vazquez).
Both authors contributed equally to this work.
1
https://doi.org/10.1016/j.biopha.2018.02.122
Received 11 January 2018; Received in revised form 19 February 2018; Accepted 23 February 2018
0753-3322/©2018ElsevierMassonSAS.Allrightsreserved.

M.E. González-Trujano et al.
Biomedicine&Pharmacotherapy101(2018)553–562
Drug discovery for pain medications aims to develop new NSAIDs
with a good analgesic effect to side effect ratio. In order to look for
therapeutic options for pain, inflammation and fever reducing adverse
drug reactions, such as damage to the digestive, kidney and cardio-
vascular systems; the search for new bioactive molecules with non-
steroidal anti-inflammatory effects that produce minimal adverse ef-
fects continues. Therefore, our present study assesses the anti-
nociceptive and/or anti-inflammatory activities of designed and syn-
thesized molecules as bioisosteres and hybrids of paracetamol,
ibuprofen and naproxen in experimental pain.
8.8 Hz), 7.84 (m, 3H, H-3, H-7, H-8) and 9.99 (s, 1H, NH) ppm. 13C NMR
(100 MHz, DMSO-d6) δ: 18.5 (CH3CH), 24.0 (CH3CO), 44.6 (CH3CH), 55.3
(-OCH3), 105.9 (C-4), 118.9 (C-6), 119.9 (C.2″, C-6″), 121.7 (C-3″, C-5″),
125.9 (C-3), 126.3 (C-2), 127.3 (C-8), 128.6 (C-7a), 129.3 (C-7), 133.6 (C-
3a), 135.4 (C-1), 137.1 (C-4″), 145.8 (C-1″), 157.4 (C-5), 168.3 (C]O) and
173.1 (OeC]O) ppm. MS (FAB+): m/z 364 (M+H)+
.
2.2.1.2. 4-(Acetylamino)phenyl
2-(R,S)-(4-isobutylphenyl)propanoate
(GUF-2).. This compound was characterized as light brown powder
with an 87% yield after purification by acid-base extraction from a 1:1
ethyl acetate: water mixture. Mp: 83.5–85.5 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 0.86 (d, 6H, (CH3)2CHCH2), 1.48 (d, 3H, CH3CH), 1.82 (m,
1H, (CH3)2CHCH2), 2.03 (s, 3H, CH3CO), 2.43 (d, 2H, (CH3)2CHCH2),
4.00 (q, 1H, CH), 6.93 (d, 2H, H-2”, H-6”, Jo = 8.8 Hz), 7.16 (d, 2H, H-
3, H-5, Jo = 8.4 Hz), 7.29 (d, 2H, H-2, H-6, Jo= 8 Hz), 7.57 (d, 2H, H-
3″, H-5″, Jo = 8.8 Hz) and 9.99 (s, 1H, NH) ppm. 13C NMR (100 MHz,
DMSO-d6) δ: 18.5 (CH3CH), 22.2 ((CH3)2CHCH2), 23.9 (CH3CO), 29.6
((CH3)2CHCH2), 44.2 ((CH3)2CHCH2), 44.3 (CH3CH), 119.9 (C-2″, C-
6″), 121.6 (C-3″, C-5″), 127.2 (C-3, C-5), 129.3 (C-2, C-6), 137.0 (C-4″),
140.0 (C-4), 145.7 (C-1″), 168.3 (C]O) and 173.0 (OeC]O) ppm. MS
2. Material and methods
2.1. Biological activity spectra prediction
The in silico biological activity spectra of the NSAIDs as bioisosteres
and hybrids of paracetamol, ibuprofen and naproxen were obtained
using the Prediction of Activity Spectra for Substances (PASS) on line
software (http://www.way2drug.com). Estimation of general biological
potential for these compounds was performed based on their structural
formula. It is known that PASS predicts more than 7000 types of bio-
logical activity with an average accuracy > 95% [14]. This program
was based on a robust analysis of structure-activity relationship from a
heterogeneous training set of about a million compounds. A biological
spectrum for a substance is the list of pharmacological activity types for
which the probability to be exposed (Pa) and not be exposed (Pi) values
are independent, and their values ranges from 0 to 1 [14].
(FAB+): m/z 340 (M+H)+
.
2.2.2. General preparation ofGUF-3 and GUF-4
(R,S)-ibuprofen and (S)-naproxen (2.2 mmol) were reacted with 3
equivalents of SOCl2 (6.5 mmol) under toluene reflux (110 °C) for 5 h to
obtain the corresponding acid chloride. Then, the last compound was
reacted with 5-aminotetrazole (2.8 mmol) via Schötten-Baumann re-
action using triethylamine (TEA, 1.3 equivalents) as a base and di-
chloromethane as the solvent. The reaction was maintained under ni-
trogen atmosphere [15].
2.2. Chemistry
All the starting materials and reagents were obtained commercially
from Sigma-Aldrich (St. Louis, MO, USA). Melting points were de-
termined on an SRS EZ Melt MPA120 automated apparatus from
Stanford Research Systems and are uncorrected. Thin layer chromato-
graphy on 2 × 5 cm pre-coated silica gel 60 F254 plates (E. Merck
KGaA, Darmstadt, Germany) visualized at 254–365 nm UV light was
used to monitor reactions.
The chemical structures of the synthesized compounds were con-
firmed based on of their spectral data (1H NMR, 13C NMR and M(S)-
FAB+). NMR studies were performed on INOVA-400 MHz instrument.
Chemical shifts (δH, δC) and coupling constant values (J) are given in
ppm and Hz, respectively. A standard reference of TMS (δH = 0, δC = 0)
in CDCl3 and DMSO-d6 as solvents was used. Mass spectra were re-
corded on a JEOL JM(S)-700 instrument (JEOL USA Inc., Peabody, MA,
USA).
2.2.2.1.
2-(R,S)-(4-Isobutylphenyl)-N-1H-tetrazol-5-ylpropanamide
(GUF-3). It is represented by a white powder with a 48% yield pur-
ification by extraction from a 1:1 ethyl acetate:water mixture. Mp:
195.5–197.5 °C. 1H NMR (400 MHz, DMSO-d6) δ: 0.82 (d, 6H,
(CH3)2CHCH2), 1.43 (d, 3H, CH3CH), 1.78 (m, 1H, (CH3)2CHCH2), 2.39
(d, 2H, (CH3)2CHCH2), 3.94 (q, 1H, CH), 7.11 (d, 2H, H-3, H-5,
Jo = 8 Hz), 7.27 (d, 2H, H-2, H-6, Jo = 8 Hz) and 12.17 (s, NH) ppm.
13C NMR (100 MHz, DMSO-d6) δ: 18.2 (CH3CH), 22.1 ((CH3)2CHCH2),
29.5 ((CH3)2CHCH2), 44.2 ((CH3)2CHCH2), 44.6 (CH3CH), 127.1 (C-3,
C-5), 129.1 (C-2, C-6), 137.8 (C-1), 140.0 (C-4), 149.8 (C-2″) and 173.1
(C]O) ppm. MS (FAB+): m/z 274 (M+H)+
.
2.2.2.1. (2S)-2-(6-Methoxy-2-naphthyl)-N-1H-tetrazol-5-ylpropana-
mide (GUF-4). It was obtained as a light brown powder with a 31%
yield after recrystallization from methanol. Mp: 253.9–255.6 °C. 1H
NMR-(400 MHz, DMSO-d6) δ: 1.54 (d, 3H, CH3CH), 3.85 (s, 3H,
-OCH3), 4.15 (q, 1H, CH), 7.16 (dd, 1H, H-6), 7.28 (d, 1H, H-4), 7.50
(dd, 3H, H-2, H-4, H-6) and 7.80 (m, 3H, H-3, H-7, H-8) ppm. 13C NMR
(100 MHz, DMSO-d6) δ: 18.1 (CH3CH), 44.8 (CH3CH), 55.2 (-OCH3),
105.8 (C-4), 118.8 (C-6), 125.8 (C-3), 126.1 (C-2), 127.0 (C-8), 128.3
(C-7a), 129.2 (C-7), 133.4 (C-3a), 135.5 (C-1), 149.8 (C-2″), 157.2 (C-5)
2.2.1. General preparation of GUF-1 and GUF-2
Synthesis of the GUF-1 and GUF-2 hybrids started from the car-
boxylic acids of (S)-naproxen and (R,S)-ibuprofen, which were reacted
with paracetamol (GUF-1 and GUF-2) through Steglich esterification.
This reaction consisted of placing the carboxylic acid NSAIDs and
phenol (paracetamol) in the presence of a catalytic amount of 4-di-
methylaminopyridine (4-DMAP) (< 10%) with stirring at 0 °C for
30 min in anhydrous chloroform (CHCl3). Then, the mixtures were re-
acted with 1.5 equivalents of dicyclohexylcarbodiimide (DCC) as a
coupling agent and chloroform as a solvent at room temperature for
30 h. The reactions were monitored by TLC analysis until they were
complete.
and 173.1 (OeC]O) ppm. MS (FAB+): m/z 298 (M + H)+
.
2.2.3. General preparation ofGUF-5 and GUF-6
Both compounds were synthesized by converting the (R,S)-ibu-
profen and (S)-naproxen carboxylic group into hydroxamic acid. The
compounds were prepared using (R,S)-ibuprofen (4.8 mmol) and (S)-
naproxen (4.3 mmol) dissolved in acetone at room temperature and
reacted with sodium bicarbonate (5.8 mmol) for 1 h to generate the
carboxylate salts. The naproxen and ibuprofen carboxylates were re-
acted with dimethyl sulfate (1.2 equivalents) for 24 h to produce the
corresponding methyl esters. The methyl esters were purified and dis-
solved in methanol. Subsequent, solid KOH (in 1:3 ratio with respect to
the initial mole of NSAIDs) and hydroxylamine hydrochloride (at a 1:2
ratio) were added. The reaction was maintained under a nitrogen at-
mosphere at room temperature until its completion.
2.2.1.1. 4-(Acetylamino)phenyl (2S)-2-(6-methoxy-2-naphthyl)propanoate
(GUF-1).. It was obtained as white crystals with a needle shape in an
89% yield after recrystallization from ethanol. Mp: 181.5–182.9 °C. 1H
NMR (400 MHz, DMSO-d6) δ: 1.58 (d, 3H, CH3CH), 2.03 (s, 3H, CH3CO),
3.87 (s, 3H, -OCH3), 4.18 (q, 1H, CH), 6.95 (d, 2H, H-2″, H-6″, Jo=
8.8 Hz), 7.17 (dd, 1H, H-6, Jm= 2.0 Hz, Jo= 9.0 Hz), 7.31 (d, 1H, H-4,
Jm= 2.4 Hz), 7.50 (dd, 1H, H-2, Jm= 1.6 Hz), 7.57 (d, 2H, H-3″, H-5″, Jo=
554

M.E. González-Trujano et al.
Biomedicine&Pharmacotherapy101(2018)553–562
2.2.3.1. [2-(R,S)-N-Hydroxy-2-[4-(2-methylpropyl)phenyl]propanamide]
(GUF-5). It was obtained as white crystals with a squamous shape with
s.s.). Naproxen, ibuprofen, paracetamol (purchased from Sigma-Aldrich,
St. Louis, MO, USA) and the GUF compounds were suspended in vehicle
(0.2% Tween 80 in s.s.). All treatments were freshly prepared before each
experiment and locally administered at a volume of 50 μL/paw 30 min
before formalin or carrageenan injection.
In the formalin test, compounds GUF 1–6, and the reference drugs
paracetamol, naproxen and ibuprofen were assayed at concentrations of
0.56, 1 and 3.2 mg/paw; whereas in the carrageenan test, the effects of
the active compounds GUF-1, GUF-2, and GUF-5 and reference drugs
were assayed at the maximal concentration (3.2 mg/paw). In the fact
that GUF-1 and GUF-2 were detected as the most active compounds,
they were chosen as candidates for molecular docking analysis.
a
30% yield after recrystallization from dichloromethane. Mp:
121.5–123 °C. 1H NMR (400 MHz, DMSO-d6) δ: 0.84 (d, 6H,
(CH3)2CHCH2), 1.31 (d, 3H, CH3CH), 1.79(m, 1H, (CH3)2CHCH2),
2.39 (d, 2H, (CH3)2CHCH2), 3.38 (q, 1H, H-2′), 7.07 (d, 2H, H-3, H-5,
Jo = 8 Hz), 7.21 (d, 2H, H-2, H-6, Jo = 8 Hz) and 10.61 (s, NH) ppm.
13C NMR (100 MHz, DMSO-d6) δ: 18.1(CH3CH), 22.1 ((CH3)2CHCH2),
29.5 ((CH3)2CHCH2), 41.7 ((CH3)2CHCH2), 44.2 (CH3CH), 126.9 (C-3,
C-5), 128.7 (C-2, C-6), 139.1 (C-1), 139.3 (C-4) and 170.4 (C]O) ppm.
MS (FAB+): m/z 222 (M + H)+
.
2.2.3.2. (2S)-N-Hydroxy-2-(6-methoxy-2-naphthyl)propanamide (GUF-
6). This compound was produced as white crystals with a squamous
shape in a 25% yield after recrystallization from chloroform. Mp:
168.5–169.5 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.42 (d, 3H, CH3CH),
3.59 (q, 1H, CH), 3.85 (s, 3H, -OCH3), 7.14 (dd, 1H, H-6, Jm = 2.8 Hz,
Jo= 9 Hz), 7.27 (d, 1H, H-4, Jm = 2.4 Hz), 7.46 (dd, 1H, H-2,
Jm = 1.2 Hz, Jo= 8.8 Hz), 7.76 (m, 3H, H-3, H-7, H-8), 8.84 (s, NH)
and 10.73 (s, NHOH) ppm. RMN 13C (100 MHz, DMSO-d6) δ: 18.2
(CH3CH), (CH3CH), 55.1 (-OCH3), 105.7 (C-4), 118.3 (C-6), 125.3 (C-
3), 126.5 (C-2, C-8), 128.3 (C-7a), 129.1 (C-7), 133.2 (C-3a), 137.0 (C-
2.4. In silico docking studies
Discovery Studio version 3.5, and Pymol version 1.0 were used for
visualization. The crystal structures of COX-1 and COX-2 were retrieved
from the PDB with the accession codes: 1EQG (resolution 2.6 Å) and
3NT1 (resolution 1.73 Å) respectively. Docking calculations were con-
ducted with AutoDock, version 4.2. The program performs several runs
in each docking experiment. Each run provides one predicted binding
mode. All water molecules and also the cocrystal ligands were removed
from the crystallographic structure. All torsions were allowed to rotate
during docking. The grid maps were generated and centered at the
crystallographic coordinates of the cocrystal compound. The grid di-
mensions were 60 × 60 × 60 points separated by 0.375 Å. The number
of docking runs was 100. After docking, all solutions were clustered
into groups with RMSD lower than 2.0 Å.
1), 157.0 (C-5) and 170.4 (C-]O) ppm. MS (FAB+): m/z 246 (M+H)+
.
2.3. Pharmacological evaluation
2.3.1. Animals
For the animal’s assays, we followed the experimental procedures
according to the Guidelines on Ethical Standards for Investigations of
Experimental Pain in Animals [16] and were in conformity with the
Norma Oficial Mexicana for the care and use of laboratory animals
(NOM-062-ZOO-1999). The animal protocol was approved by the local
Animal Ethics Committee (Instituto Nacional de Psiquiatría Ramón de
la Fuente Muñíz, México City). Female Wistar rats weighing 160–180 g
[Crl(WI)fBR] were housed in a temperature- and light-controlled room
under a 12-h light/12-h dark cycle (lights on at 7:00 A.M.) with water
and standard pellets ad libitum. The groups consisted of at least six rats
for all the experimental procedures. Each animal was used only one
time, and after the experiment, it was sacrificed using CO2.
2.4.1. Docking validation
The molecular docking protocols were validated by re-docking of
cocrystal ligands into the active site of the structure of each protein
target: COX-1 (ibuprofen), COX-2 (naproxen). The root mean square
deviation (RMSD) between the co-crystal ligand and the docked struc-
ture was less than 2.0 Å. This value indicates that the parameters for
docking simulations agree in reproducing orientation and conformation
in the X-ray crystal structure of enzyme and receptors.
2.4.2. Statistical analysis
2.3.2. Antinociceptive activity in the formalin test
Temporal courses were built to obtain the antinociceptive or anti-
inflammatory responses with respect to time. The percentage activity
was obtained with the area under the curve (AUC) from the temporal
courses using the trapezoidal rule. We used a linear logarithmic activity
with 95% confidence intervals to determine the ED25, as was it calcu-
lated with a least-squares linear regression according to Tallarida [20].
One-way analysis of variance (ANOVA) followed by Tukey’s test was
used to compare the differences among treatments. Differences were
considered statistically significant when P < 0.05. The statistical
analysis was performed using Graphpad Prism® version 5.0 for Win-
dows (Graphpad Software, San Diego, CA, USA).
Nociception was induced in the formalin test in rats as previously
described [17] under experimental conditions [18]. Nociceptive beha-
viors were quantified as the numbers of flinches, withdrawal or flexing
of the injected paw (50 μL) over the course of 1 h.
2.3.3. Anti-inflammatory activity using carrageenan paw edema test
Rats received an intraplantar injection of 1% carrageenan (50 μL)
into the right hind paw [19]. Paw volume was measured by means of a
volume displacement method using a plethysmometer (plethysmometer
7150, UGO Basile, Italy) immediately prior to the carrageenan injection
and thereafter at 2 and 4 h. Edema was expressed as the increase in paw
volume (mL) after carrageenan injection relative to the pre-injection
value for each animal. The percent inhibition was calculated as follows:
3. Results
a − x
b − y
⎛
⎞
⎟
% Anti − inflammation = 1 −
× 100
⎜
3.1. PASS online
⎝
⎠
where ‘x’ and ‘a’ are the mean paw volume of the rats in the test group
before and after carrageenan injection, respectively, and ‘y’ and ‘b’ are
the mean paw volume of rat before and after carrageenan injection in
the control group, respectively.
Table 1 presents the PASS online data for the antinociceptive, anti-
inflammatory, and antipyretic activities of GUF1 to 6. These data show
that GUF-1 and GUF-6 presented moderate, in comparison to high of
GUF-2, antinociceptive, anti-inflammatory and antipyretic activities;
while GUF-3 and GUF-4 showed only moderate anti-inflammatory ac-
tivity. The activity probability (Pa) was higher than the inactivity
probability (Pi) in all the compounds. According to these results, GUF-
1–6 were synthesized.
2.3.4. Experimental design
One percent formalin was dissolved in saline solution (s.s., 0.9% NaCl),
and 1% carrageenan was suspended in 0.5% carboxymethylcellulose (in
555

M.E. González-Trujano et al.
Biomedicine&Pharmacotherapy101(2018)553–562
and GUF-2 inhibited both phases of the formalin test, we decided to
Table 1
Predictive antinociceptive, anti-inflammatory, and antipyretic values for GUF 1–6 de-
examine the molecular docking of both compounds.
termined by PASS.14
Compound
Antinociceptive
Anti-inflammatory
Antipyretic
3.4. Docking studies
Pa
Pi
Pa
Pi
Pa
Pi
The biological assays showed that GUF-1, GUF-2, and GUF-5 pos-
sess antinociceptive activity; thus, an in silico analysis was used to
evaluate the molecular interactions between these compounds and the
ligand binding site of each COX isoform. To validate the computational
analytical method, we used the RSMD variation (less than 2Å) of ibu-
profen and naproxen individually into the active site of both isoforms.
On the other hand, we found that both reference drugs have polar in-
teractions with the COX catalytic site; they act as hydrogen bond ac-
ceptors with Arg-120 and Tyr-355, and have long-range polar interac-
tions with Tyr-385 and Ser-530 and non-polar interactions with Val-
349, Trp-387, Phe-518, Ile/Val-523 and Ala-527.
GUF-1
GUF-2
GUF-3
GUF-4
GUF-5
GUF-6
0.617
0.673
0.257
0.392
0.379
0.564
0.022
0.015
0.177
0.096
0.095
0.030
0.741
0.745
0.668
0.658
0.523
0.684
0.009
0.009
0.015
0.017
0.046
0.014
0.922
0.924
0.200
0.232
0.448
0.863
0.003
0.003
0.152
0.118
0.022
0.004
Pa = activity probability.
Pi = inactivity probability.
3.2. Chemistry
In the pharmacological evaluation, GUF-1 displayed antinociceptive
and anti-inflammatory properties; thus, molecular docking analysis was
performed to identify important interactions to potentially increase the
biological activity. The interactions between GUF-1 in the COX-1 ligand
binding site compared to the reference drugs decreased, leading to an
interaction with Tyr-385 where the methoxy group acts as a hydrogen
bond acceptor, and another long-range polar interaction where Ser-530
acts as a hydrogen bond donor. The phenyl ring makes a π-cation in-
teraction with Arg-120, and the molecule has polar interactions with
Ser-353, Tyr-348 and 355 and non polar interactions with Val-349, Trp-
387, Phe-518, Ala-527 and Ile-523. Figs. 7–10 depict the corresponding
amino acid residues at 4.5 Å in a 2D interaction map generated with the
program Molecular Operating Environment [21].
Moreover, the conformation adopted by GUF-1 was different. In
COX-2, the compound made other molecular interactions with the li-
gand binding site that included an N-acetyl group acting as a hydrogen
bond acceptor with His-90 and Leu-352. The compound forms polar
interactions with Arg-120, Ser-353, Tyr-355, Gly-526, and Ser-530 and
non-polar interactions with Val-116, 349 and 523; Leu-359; Phe-518;
Ala-527 and Leu-531 (Fig. 8).
Through Steglich esterification, the synthesis of the two hybrids:
paracetamol-naproxen (GUF-1) and paracetamol-ibuprofen (GUF-2)
was performed. Other NSAID-aminotetrazoles (GUF-3 and GUF-4) and
NSAID-hydroxamic acids (GUF-5 and GUF-6) bioisosteres were also
synthesized. Figs. 1–3 show the synthetic route and chemical structures
of GUF-1 to GUF-6. All compounds were characterized based on their
spectral data (1H and 13C NMR, as well as mass spectra FAB-MS).
3.3. Pharmacology
In this study, typical NSAID administration was used as a positive
control for comparison with the new GUF compounds. Fig. 4 shows the
typical antinociception produced by local injection of paracetamol,
naproxen, and ibuprofen. In all the cases, there are not antinociceptive
responses in the neurogenic stage (phase 1 from 0–10 min). Para-
cetamol produced a concentration-dependent antinociceptive response
after a short quiescent period (15 min), while naproxen and ibuprofen
showed antinociception in a non-concentration-dependent manner in
the inflammatory stage (phase 2 from 15–60 min).
All the synthesized GUFs were evaluated using the 1% formalin test;
GUF-3, GUF-4, and GUF-6 did not show any antinociceptive effects
compared with the vehicle control (data not shown). Fig. 5 shows an-
tinociceptive responses of GUF-1 and GUF-2 in phases 1 and 2, GUF-5
reduced formalin-induced flinching during phase 2 in a concentration-
dependent fashion (P < 0.05) but not in phase 1. All the GUFs (1, 2
and 5) showed more that 50% antinociceptive response at 3.2 mg/paw
The compound GUF-2 did not show several polar interactions with
the COX-1 ligand binding center. The steric space and conformation
assumed by the compound prevented donor and hydrogen bond ac-
ceptors from aligning with the polar amino acids such as the polar
amino acids in the catalytic center are in contact with the non-polar
parts of GUF-2, such as in the case of the phenyl interacting with Tyr-
348 and Ser-530 and the isobutyl in close contact with Ser-353 and Tyr-
355. Similarly, the N-acetyl group is surrounded by hydrophobic amino
acids including Phe-205, 209 and 381, and the ester is surrounded by
Leu-352, Ile-523, Ala-527 and Leu-531 (Fig. 9).
In the COX-2 isoform, the GUF-2 N-acetyl carbonyl oxygen made
polar contacts with His-90 and similar interactions with Leu-352 and
Ser-353. The GUF-2 amino group formed interactions that supported
the structure via Leu-352. The phenyl ring made a π-hydrogen inter-
action with Leu-531. Long-range polar interactions were made with
Tyr-355, Ser-353 and 355, while non-polar interactions were formed
with Val-116 and 349 (Fig. 10).
concentration with a similar potency (GUF-1, ED25= 0.9
paw; GUF-2, ED25 0.7 0.3 mg/paw, and GUF-5, ED25
0.5 0.5 mg/paw).
Since the higher nociceptive response was obtained at 3.2 mg/paw
0.3 mg/
=
=
administration, it was also used to analyze the effects of the new GUFs
in the inflammatory process induced by carrageenan. Fig. 6 shows that
GUF-1, GUF-2, and GUF-5 demonstrated anti-inflammatory responses
resembling the effect of naproxen and ibuprofen, while paracetamol did
not show anti-inflammatory response. Thus, only these three com-
pounds could be useful for treating inflammatory pain. Due to GUF-1
Fig. 1. Formation of ibuprofen, naproxen, and paracetamol heterodimers (GUF-1 and GUF-2).
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Biomedicine&Pharmacotherapy101(2018)553–562
Fig. 2. Synthesis of bioisosteres of ibuprofen and naproxen with tetrazole (GUF-3 and GUF-4).
Derived from its chemical structure, the conformation of GU F-2 in
the COX-2 ligand binding center demonstrated multiple interactions
that could displace the coupling equilibrium towards this isoform; this
paracetamol. In COX-2, GUF-1 and GUF-2 are in an inverted "V" con-
formation, meaning that the respective parts to the aryl propionic
NSAIDs are aligned and that the compounds matched the binding or-
ientation of paracetamol.
allowed
a better selectivity in the antinociceptive and anti-in-
flammatory responses of GUF-2 via COX-2 inhibition minimizing the
ulcerogenic effects and the risk of cardiotoxicity. In contrast, GUF-2
interactions with COX-1 were almost non-existent. No direct polar
contacts were generated among the amino acids surrounding the mo-
lecule and the conformation; it is assumed that there is no facilitation in
the interaction with the catalytic protein center. Based on the overlap of
the ligands in both isoforms, the molecular accommodation of GUF-1
and GUF-2 in COX-1 showed greater depth in the catalytic cavity, as-
suming an "inverted V" orientation, the reverse of the orientation of
Finally, the Gibbs free energies of the most active compounds were
calculated. The Gibbs free energies of the NSAID reference drugs had a
higher negative value for the COX-2 conformation, so less energy is
needed to stabilize the drugs at the ligand binding center, this same
assertion is congruent with the calculated affinity constant (Ki) when
the selectivity index > 1. For GUF-1, the free energy in isoform 1 was
larger than that of the reference drugs, while it was lower than the
reference drugs in isoform 2, meaning that greater energy is required to
stabilize the compound in isoform 1 compared with isoform 2 (Table 2).
Fig. 3. Synthesis of bioisosteres of ibuprofen and naproxen with hydroxamic acid (GUF-5 and GUF-6).
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Biomedicine&Pharmacotherapy101(2018)553–562
Fig. 4. Antinociceptive effects of paracetamol, naproxen and ibuprofen at 0.56, 1 and 3.2 mg/paw using the 1% formalin test. The vehicle group (0.9% NaCl) showed the total nociceptive
effect of 1% formalin in the dorsal paw injection. The left panels show the time course curves for (A) paracetamol, (C) naproxen and (E) ibuprofen. The right panels show the AUC of the
corresponding temporal course curves for (B) paracetamol, (D) naproxen and (F) ibuprofen in the second phase of the formalin test in rats. Data are presented as the mean
SEM, n = 6.
*P < 0.05, ANOVA followed by Tukey's test.
Although the affinity constant (Ki) for GUF-1 in COX-2 is the
average of the reference drugs, GUF-2 has six-fold the affinity of GUF-1
to inhibit COX-1 and requires a greater concentration to generate the
same effect. Thus, the selectivity coefficient of GUF-1 was 40.77 times
more selective for COX-2 than COX-1.
4. Discussion
In this study, hybrid compounds and bioisosteres of some NSAIDs
were designed. Bioisosteres are chemical substituents or groups with
similar physicochemical properties which produce broadly similar
pharmacological properties to another drug [22]. For this study,
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Biomedicine&Pharmacotherapy101(2018)553–562
Fig. 5. Antinociceptive effects of GUF-1, GUF-2, and GUF-5 at 0.56, 1 and 3.2 mg/paw concentrations in the 1% formalin test. The vehicle group (0.9% NaCl) showed the total
nociceptive effect of 1% formalin in the dorsal paw injection. Left panels show the time course curves for (A) GUF-1, (C) GUF-2, and (E) GUF-5. The right panels show the AUC of the
respective temporal course for (B) GUF-1, (D) GUF-2, and (F) GUF-3 in phase 2 of the formalin test in rats. Data are presented as the mean
SEM, n = 6. *P < 0.05, ANOVA followed
by Tukey's test.
ibuprofen was selected because it was the first arylpropionic acid de-
rivative to be used in its general form as racemic mixture, the case of
naproxen was due to its longer half-life than other drugs with structural
and functional similarities and paracetamol that is because it is an ef-
fective compound that can be used as an analgesic-antipyretic drug. It is
well-tolerated and does not generate many of the side effects of most
NSAIDs. For the specific case of paracetamol, it has an arylhydroxyl
group and more acidic characteristics than water due to the resonance
stability of the phenoxide ion. The effect of this resonance is the dis-
tribution of the anion charge over the entire molecule instead of having
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Biomedicine&Pharmacotherapy101(2018)553–562
Fig. 6. Anti-inflammatory effects elicited by the subcutaneous administration ofGUFs and NSAIDs in the carrageenan-induced edema paw test. Rats were administered 3.2 mg/paw of (A)
GUF-1 (Δ), GUF-2 (□) and GUF-5 (∇) or (B) paracetamol (▲), ibuprofen (■) and naproxen (▼) 30 min before carrageenan injection. Data are expressed as the inflammation (%) as a
function of time. The vehicle group (0.9% NaCl) showed the total inflammatory effect of 1% carrageenan injection. Data are presented as the mean
ANOVA followed by Bonferroni's test. In the plot, asterisks indicate significant differences among NSAIDs and GUFs or the vehicle.
SEM, n = 6. *P < 0.05, two-way
Fig. 7. (A) 3D binding model of GUF-1 (green) into the ligand binding site of COX-1. (B) 2D interaction map.
Fig. 8. (A) 3D binding model of GUF-1 (green) into the ligand binding site of COX-2. (B) 2D interaction map.
it concentrated on a particular atom. This increases the ability of this
compound to react. Arylpropionic NSAIDs have acidic properties be-
cause they have a carboxylic acid group; the two oxygen atoms are
electronegative and attract electrons from the hydroxyl group hydrogen
atom, which weakens the bond, and can produce a heterolytic rupture
in certain conditions that yields the corresponding proton and leaves
the rest of the molecule with a negative charge. This charge symme-
trically translocates between the two oxygen atoms, yielding carbon-
oxygen bonds with partial double bond characteristics. Thus, we
created aryl propionic drugs by synthesizing esters of ibuprofen and
naproxen with paracetamol to mask the acidic characteristics of the
NSAIDs. The non-classical bioisosteres of ibuprofen and naproxen were
considered for replacing the carboxylic acid functional group with the
acylaminotetrazole group.
Our data showed that an acute 1% formalin injection produces two
phases to characterize the nociceptive process in rats. The formalin test
is a tonic model of pain that results from formalin-induced local tissue
injury. It is a useful model for screening novel compounds because it
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Biomedicine&Pharmacotherapy101(2018)553–562
Fig. 9. (A) 3D binding model of GUF-2 (green) into the ligand binding site of COX-1. (B) 2D interaction map.
Fig. 10. (A) 3D binding model of GUF-2 (green) into the ligand binding site of COX-2. (B) 2D interaction map.
GUF-2 can be attributed to their interactions with the COX ligand
binding center. Other authors have also demonstrated anti-in-
flammatory activities by synthesizing naproxen and ibuprofen hybrids
using natural products such as terpenes (ferruginol, imbricatolic acid
and oleanolic acid) with naproxen or ibuprofen [26]. The β-boswellic
acid and the 11-keto-β-boswellic acid from Boswellia serrata resin were
subjected to Steglich esterification with ibuprofen, naproxen, diclo-
fenac and indomethacin; this led to hybrid compounds with increased
antiarthritic and anti-inflammatory effects compared with NSAIDs [27].
Our data using Steglich esterification [28] with different NSAIDs
showed also that it is possible to increase the potential antinociceptive
response.
Table 2
Gibbs free energy and affinity constant of the reference drugs and GUF-1 and GUF-2
determined in silico.
Compound COX-1
COX-2
In silico selectivity
index Ki (COX-1/COX-
Ki (μM) 2)
ΔG°
Ki (μM) ΔG°
(Kcal/
mol)
(Kcal/
mol)
Ibuprofen −8.58
Naproxen
GUF-1
0.509
0.257
8.4
−8.91
−9.38
−9.12
−8.0
0.291
0.133
0.206
1.38
1.74
1.93
40.77
0.87
−8.99
−6.92
−8.07
GUF-2
1.21
5. Conclusion
encompasses the inflammatory, neurogenic, and central mechanisms of
nociception. Subcutaneous injection of 1% formalin into the hind paw
produced a typical pattern of flinching behaviors characterized by a
biphasic time-course from a quiescent interphase. Phase 1 of the noci-
ceptive response began immediately after formalin administration and
then gradually decreased over approximately 10 min. Phase 2 began
approximately 15 min after formalin administration and lasted around
60 min after formalin injection [23]. Our data using standard com-
pounds agreed with the literature showing that NSAIDs only demon-
strated antinociceptive activity in phase 2 [24,25]. It is well-known that
the nociceptive process depends on the nociceptor activation in phase 1
and that prostaglandins do not play a key role during this phase. The
late phase appears to be the inflammatory response with inflammatory
pain that can be inhibited by anti-inflammatory drugs.
In this study, we showed that three compounds: hybrids GUF-1,
GUF-2, and the bioisostere GUF-5 showed greater antinociceptive ef-
ficacy than their precursors naproxen, ibuprofen, and paracetamol,
used mainly as traditional NSAIDs.
Conflict of Interests
The authors declared that there is no conflict of interests regarding
the publication of this paper.
Acknowledgements
The molecular docking observations can be correlated with the
biological activity. GUF-1, GUF-2 and GUF-5 showed better anti-
nociceptive activity in phase 2; these results for hybrids GUF-1 and
We thank the Facultad de Farmacia internal grant for providing
some research materials for the study. This work was partially sup-
ported by Consejo Nacional
de Ciencia
y
Tecnología
561

M.E. González-Trujano et al.
Biomedicine&Pharmacotherapy101(2018)553–562
(CONACYT226454/256448) and Instituto Nacional de Psiquiatria (INP
NC123280.0/NC17073.0). We are also grateful to Profr. Marissa
González for proof-reading this manuscript.The work was taken in part
from the B.Pharm Thesis of G. Uribe-Figueroa.
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