Benzofuranones as potential antinociceptive agents: Structure-activity relationships

Article abstract of DOI:10.1016/j.ejmech.2012.08.015

This work evaluates the antinociceptive properties of benzofuranones using chemically induced models of pain and the hot plate test. All the compounds exhibited significant antinociceptive activity, with 3-[2-(4-chlorophenyl)-2- oxoetil]-2-benzofuran-1(3H)-one (3d) being the most active. According to the application of the Topliss method, the 2π-π² parameter was the preponderant one, indicating that the hydrophobicity (π) seems to be more involved in the antinociceptive activity. Based on the table of other possible substituents proposed by Topliss, three derived from compound 3d were tested. 3-[2-(3-methoxyphenyl)-2-oxoetil]-2-benzofuran-1(3H)-one (3g) showed greater antinociceptive activity with better pharmacokinetic properties predicted. These results show the efficiency of the Topliss Method as a research tool for the discovery of potential candidate molecules for a new antinociceptive drug.

Full text of DOI:10.1016/j.ejmech.2012.08.015

European Journal of Medicinal Chemistry 56 (2012) 120e126
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzofuranones as potential antinociceptive agents: Structureeactivity
relationships
,
,b
Cleiton José Gonçalves a, Andrey Sávio Lenoir a, Pâmela Padaratz b, Rogério Corrêa a b, Rivaldo Niero a
,
,
,b,
*
Valdir Cechinel-Filho a b, Fátima de Campos Buzzi a
a Núcleo de Investigações Químico-Farmacêuticas (NIQFAR)/CCS, Universidade do Vale do Itajaí-UNIVALI, Caixa Postal 360, CEP 88.302-202 Itajaí, Santa Catarina, Brazil
b Programa de Pós-graduação em Ciências Farmacêuticas, Universidade do Vale do Itajaí-UNIVALI, 88.302-202 Itajaí, Santa Catarina, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This work evaluates the antinociceptive properties of benzofuranones using chemically induced models
of pain and the hot plate test. All the compounds exhibited significant antinociceptive activity, with 3-[2-(4-
chlorophenyl)-2-oxoetil]-2-benzofuran-1(3H)-one (3d) being the most active. According to the application
Received 19 May 2012
Received in revised form
8 August 2012
Accepted 9 August 2012
Available online 16 August 2012
of the Topliss method, the 2
) seems to be more involved in the antinociceptive activity. Based on the table of other possible
pep
2 parameter was the preponderant one, indicating that the hydrophobicity
(p
substituents proposed by Topliss, three derived from compound 3d were tested. 3-[2-(3-methoxyphenyl)-
2-oxoetil]-2-benzofuran-1(3H)-one (3g) showed greater antinociceptive activity with better pharmacoki-
netic properties predicted. These results show the efficiency of the Topliss Method as a research tool for the
discovery of potential candidate molecules for a new antinociceptive drug.
Keywords:
Benzofuranone
Antinociceptive properties
Topliss method
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
than the original chalcone and some clinically used drugs [14]. In
the present study, we have extended our observations, synthesizing
Chalcones are a group of plant-derived polyphenolic compounds
belonging to the flavonoids family that can also be obtained by
synthetic route. This class has a wide variety of biological properties,
including antinociceptive and anti-inflammatory [1e3], antitu-
moral [4] antibacterial and antifungal [3,5], and antileishmanial [6]
properties, among others [7e9]. Chalcone-containing plants have
long been used as medical treatments in Asia, Africa and South
America. Several pure chalcones were approved for clinical use or
tested in humans, such as Metochalcone, marketed as a choleretic
drug, and Sofalcone, marketed as an anti-ulcer and mucoprotective
drug [10,11]. Benzofuranones are a group of substances that can be
obtained by synthesis from several starting products, including
chalcones. Several compounds belonging to this class exhibit
a broad spectrum of biological properties, such as antinociceptive,
cytotoxic, antibacterial and antioxidant properties [12e15]. In
previous studies, we have demonstrated that a new benzofuranone,
denoted 3-[2-(2-hydroxy-4, 6-dimethoxyphenyl)-2-oxoethyl]-2-
benzofuran-1(3H)-one, was more potent as an antinociceptive
some benzofuranones and evaluating their possible antinociceptive
effects. In addition, the Topliss method was used to obtain more
potent substances.
2. Materials and methods
2.1. Chemistry
The melting points were determined by a Microquímica WG
APF-301 apparatus, and are uncorrected. IR spectra were recorded
in KBr using a Perkin Elmer 16 PC-FT spectrometer. The 1H NMR and
13C spectra were recorded in the solvent indicated, with a Bruker
WM 300 MHz spectrometer, with TMS as internal standard. The
purity of the synthesized substances was monitored by thin-layer
chromatography (TLC) using silica gel 60 F254 pre-coated
aluminum sheets (MerckÒ) with several solvent systems of
different polarities.
The compounds were synthesized from carboxybenzaldehyde
and different acetophenones (Fig. 1). Initially the acetophenones
(0.92 mmol) were dissolved in ethanol (25 ml) and mixed in
a solution of NaOH 50% (p/v; 2.5 mmol). The carboxybenzaldehyde
(0.95 mmol) was then added. The reaction was heated for 14 h, then
acidified with HCl at 50% until complete precipitation. The product
was recrystallized in ethanol (10 ml) and filtered in a Buchner
* Corresponding author. Núcleo de Investigações Químico-Farmacêuticas
(NIQFAR)/CCS, Universidade do Vale do Itajaí (UNIVALI), Caixa Postal 360, CEP
88302-202, Itajaí, Santa Catarina, Brazil. Tel.: þ55 47 3341 7855; fax: þ55 47
3341 7601.
E-mail address: fcamposbuzzi@univali.br (F.de Campos Buzzi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.015

C.J. Gonçalves et al. / European Journal of Medicinal Chemistry 56 (2012) 120e126
121
funnel. The residue was purified on a silica gel column (eluent:
hexaneeethyl acetate).
NMR (300 MHz, CDCl3,
d
ppm): 7.89 (d, 1H, J ¼ 9.0 Hz, H-50), 7.66
(dd, 1H, J ¼ 6.0, 2.0 Hz, H-60), 7.53 (d, 1H, J ¼ 2.0 Hz, H-20), 7.53e7.59
(m, 4H, H-ring-B), 6.18 (dd, 1H, J ¼ 12.0; 6.0 Hz, H-8), 3.96 (s, 6H, 2ꢂ
2.1.1. 3-(2-Oxo-2-phenylethyl)-2-benzofuran-1(3H)-one (3a)
Colorless crystals, yield 53%, mp: 146.5e147.5 ꢀC, IR (KBr, cmꢁ1):
3062 (CHeAr), 1771 (C]OeC), 1682 (C]O) and 1212 (CeO). 1H
OCH3), 3.75 (dd, 1H, J ¼ 15.0; 6.0 Hz, H-9
a
), 3.36 (dd, 1H, J ¼ 15.0;
6.0 Hz, H-9b d ppm): 194.4 (C-10), 170.1
). 13C NMR (75 MHz, CDCl3,
(C-7), 153.9e110.3 (C]C-ring A and B), 76.4 (C-8), 56.1 (2ꢂ OCH3),
43.2 (C-9). Anal. Calc. for C18H16O5: C, 69.22; H, 5.16. Found: C,
69.32; H, 5.10.
NMR (300 MHz, DMSO-d6,
B), 6.11 (dd, 1H, J ¼ 6.0, 3.0 Hz, H-8), 3.81 (dd, 1H, J ¼ 12.0; 6.0 Hz;
H-9
), 3.78 (dd, 1H, J ¼ 12.0; 6.0 Hz; H-9
). 13C NMR (75 MHz,
DMSO-d6, ppm): 196.5 (C-10), 169.9 (C-7), 149.9e122.9 (C]C-ring
d ppm): 7.51e8.01 (m, 9H, H-ring A and
a
b
d
2.1.7. 3-[2-(3-Methoxyphenyl)-2-oxoethyl]-2-benzofuran-1(3H)-
one (3g)
A and B), 77.2 (C-8), 42.7 (C-9), Anal. Calc. for C16H12O3: C, 76.18; H,
4.79. Found: C, 76.15; H, 4.81.
White powder, yield 57%, mp: 134.2e135.8 ꢀC. IR (KBr, cmꢁ1):
1765 (C]OeC), 1682 (C]O) and 1255 (CeO). 1H NMR (300 MHz,
2.1.2. 3-[2-(4-Metoxyphenyl)-2-oxoethyl]-2-benzofuran-1(3H)-
one (3b)
CDCl3,
d
ppm): 7.92 (d, 1H, J ¼ 7.5 Hz, H-40), 7.66 (dd, 1H, J ¼ 7.5;
2.0 Hz, H-60), 7.36e7.56 (m, 4H, H-ring-B), 7.50 (d, 1H, J ¼ 2.0 Hz,
Colorless, yield 68%; mp: 121e122 5 ꢀC, IR (KBr, cmꢁ1): 1759 (C]
OeC), 1676 (C]O) and 1216 (CeO). 1H NMR (300 MHz, CDCl3,
H-20), 6.15 (dd, 1H, J ¼ 9.0; 2.0 Hz, H-8), 3.86 (s, 3H, OCH3), 3.74 (dd,
1H, J ¼ 12.0; 6.0 Hz, H-9
a
), 3.38 (dd, 1H, J ¼ 12.0; 6.0 Hz, H-9
b C
). 13
d
ppm): 6.92 (d, 2H, J ¼ 9.0 Hz, H-20 and H-60), 7.94 (d, 2H, J ¼ 9.0 Hz,
NMR (75 MHz, CDCl3, d ppm): 195.8 (C-10), 170.1 (C-7), 159.9e112.2
H-30 and H-50), 7.65e7.50 (m, 4H, H-ring-B), 6.15 (dd, 1H, J ¼ 12.0;
(C]C-ring A and B), 76.6 (C-8), 55.4 (OCH3), 43.7 (C-9). Anal. Calc.
for C17H14O4: C, 72.33; H, 5.00. Found: C, 72.30; H, 5.35.
6.0 Hz, H-8), 3.71 (dd, 1H, J ¼ 15.0; 6.0 Hz, H-9
a
), 3.34 (dd, 1H,
J ¼ 15.0; 6.0 Hz, H-9
b
), 3.87 (s, 3H, OeCH3). 13C NMR (75 MHz,
CDCl3, ppm): 194.3 (C-10), 170.1 (C-7), 149.8e122.8 (C]C-ring A
d
2.1.8. 3-[2-(4-Bromophenyl)-2-oxoethyl]-2-benzofuran-1(3H)-
one (3h)
and B), 76.5 (C-8), 55.4 (OCH3), 43.2 (C-9). Anal. Calc. for C17H14O4:
C, 72.33; H, 5.00. Found: C, 72.31; H, 5.03.
White powder, yield 43%, mp: 147.0e149.9 ꢀC. IR (KBr, cmꢁ1):
1749 (C]OeC), 1682 (C]O) and 1215.92 (CeO). 1H NMR (300 MHz,
2.1.3. 3-[2-(4-Methylphenyl)-2-oxoethyl]-2-benzofuran-1(3H)-
one (3c)
CDCl3,
d
ppm): 7.82 (dd, 2H, J ¼ 9.0 Hz, H-20 and H-60), 7.98 (dd, 2H,
J ¼ 9.0 Hz, H-30 and H-50), 7.58e7.85 (m, 4H, H-ring-B), 6.15 (dd, 1H,
Colorless, yield 64%, mp: 149.1e150.1 ꢀC, IR (KBr, cmꢁ1): 1761
(C]OeC), 1673 (C]O) and 1295 (CeO). 1H NMR (300 MHz, CDCl3,
J ¼ 18.0; 9.0 Hz, H-8), 3.76 (d, 2H, J ¼ 9.0 Hz, H-9
a and H-9b C
). 13
NMR (75 MHz, CDCl3,
d ppm): 195.4 (C-10), 169.6 (C-7),150.2e123.1
d
ppm): 7.34 (d, 2H, J ¼ 9.0 Hz, H-20 and H-60), 7.95 (d, 2H, J ¼ 9.0 Hz,
(C]C-ring and B), 77.0 (C-8), 43.3 (C-9). Anal. Calc. for
A
H-30 and H-50), 7.41e7.85 (m, 4H, H-ring-B), 6.15 (dd, 1H, J ¼ 12.0;
C16H11BrO3: C, 58.03; H, 3.35. Found: C, 58.11; H, 3.33.
6.0 Hz, H-8), 3.72 (dd, 1H, J ¼ 12.0; 6.0 Hz, H-9
a), 3.74 (dd, 1H,
J ¼ 12.0; 6.0 Hz, H-9
b
), 2.40 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3,
2.2. Biological assays
d
ppm): 196.0 (C-10), 170.1 (C-7), 150.7e123.3 (C]C-ring A and B),
77.7 (C-8), 43.6 (AreCH3), 43.4 (C-9). Anal. Calc. for C17H14O3: C,
76.68; H, 5.30. Found: C, 76.65; H, 5.25.
2.2.1. Animals
Male Swiss mice (25e35 g) were used, housed at 22 ꢃ 2 ꢀC
under a 12 h light/12 h dark cycle, with access to food and water ad
libitum. The experiments were performed during the light phase of
the cycle. The animals were acclimatized to the laboratory for at
least 2 h before testing, and were used once throughout the
experiments. All the experiments reported in this study were
carried out in accordance with the current guidelines for the care of
laboratory animals and the ethical guidelines for investigation of
experimental pain in conscious animals [16]. The number of mice
(6e8 per group) and intensity of noxious stimuli used were the
minimum necessary to demonstrate consistent effects of the drug
treatments.
2.1.4. 3-[2-(4-Chlorophenyl)-2-oxoethyl]-2-benzofuran-1(3H)-
one (3d)
White powder, yield 45%, mp: 147.5e148.6 ꢀC, IR (KBr, cmꢁ1):
3061 (CHeAr), 1754 (C]OeC), 1679 (C]O) and 1217 (CeO). 1H
NMR (300 MHz, CDCl3,
d
ppm): 7.45 (d, 4H, J ¼ 9.0 Hz, H-20 and
H-60), 7.90 (d, 2H, J ¼ 9.0 Hz, H-30 and H-50), 7.58e7.67 (m, 4H,
H-ring-B), 6.10 (dd, 1H, J ¼ 12.0; 6.0 Hz, H-8), 3.71 (dd, 1H, J ¼ 15.0;
6.0 Hz, H-9
(75 MHz, CDCl3,
a
), 3.35 (dd, 1H, J ¼ 15.0; 6.0 Hz, H-9
b
). 13C NMR
d
ppm): 194.8 (C-10), 170.0 (C-7), 149.5e122.7 (C]
C-ring A and B), 76.6 (C-8), 43.6 (C-9). Anal. Calc. for C16H11ClO3: C,
67.06; H, 3.87. Found: C, 67.12; H, 3.82.
2.2.2. Drugs
2.1.5. 3-[2-(3,4-Dichlorophenyl)-2-oxoethyl]-2-benzofuran-1(3H)-
one (3e)
The following substances were used: acetic acid, formalin,
-glutamic acid, capsaicin (Calbiochem, San Diego, CA, USA),
L
White powder, yield 78%, mp: 192.3e192.4 ꢀC, IR (KBr, cmꢁ1):
morphine hydrochloride and naloxone (Merck, Darmstadt,
Germany). All synthetic compounds, as well as the reference drugs,
were dissolved in Tween 80 (E. Merck), plus 0.9% of NaCl solution,
with the exception of the capsaicin, which was dissolved in ethanol.
The final concentration of Tween 80 and ethanol did not exceed 5%
and did not cause any effect per se.
1751 (C]OeC), 1682 (C]O) and 1215 (CeO). 1H NMR (300 MHz,
CDCl3,
d
ppm): 7.95 (d, 2H, J ¼ 9.0, H-30 and H-50), 7.85 (d, 2H,
J ¼ 9.0 Hz, H-20 and H-60), 7.62e7.83 (m, 4H, H-ring-B), 6.10 (dd, 1H,
J ¼ 12.0; 6.0 Hz, H-8), 3.80 (dd, 1H, J ¼ 15.0; 6.0 Hz, H-9
a), 3.86 (dd,
1H, J ¼ 15.0; 6.0 Hz, H-9
b d ppm): 194.8
). 13C NMR (75 MHz, CDCl3,
(C-10),169.7 (C-7),150.5e122.2 (C]C-ring A and B), 76.8 (C-8), 43.8
(C-9). Anal. Calc. for C16H10Cl2O3: C, 59.84; H, 3.12. Found: C, 59.82;
H, 3.12.
2.2.3. Writhing test
Abdominal constriction was induced in mice by intraperitoneal
injection of acetic acid (0.6%), as described by Collier et al. [17] with
minor modifications. The animals were pre-treated intraperitone-
ally (0.5 at 30 mg kgꢁ1, 30 min before) for all compounds, and orally
(30, 60 and 100 mg kgꢁ1,1 h before) with compound 3g. The control
animals received a similar volume of saline solution (10 ml kgꢁ1).
2.1.6. 3-[2-(3,4-Dimethoxyphenyl)-2-oxoethyl]-2-benzofuran-
1(3H)-one (3f)
White powder, yield 55%, mp: 152.0e152.7 ꢀC. IR (KBr, cmꢁ1):
3087 (CHeAr), 1760 (C]OeC), 1670 (C]O) and 1259 (CeO). 1H

122
C.J. Gonçalves et al. / European Journal of Medicinal Chemistry 56 (2012) 120e126
O
O
O
O
2'
3'
O
7
10
H
O
1'
NaOH
CH3
A
8
4'
6
B
9
A
+
6'
1
2
5
4
5'
EtOH/H2O
B
3
X
OH
X
2
1
X= H(3a), 4-OCH3(3b), 4-CH3(3c), 4-Cl(3d), 3,4-Cl2(3e), 3,4-OCH3(3f), 3-OCH3(3g), 4-Br(3h)
Fig. 1. General route for preparation of benzofuranone derivatives.
The number of abdominal constrictions (full extension of both hind
paws) was cumulatively counted over a period of 20 min. Anti-
nociceptive activity was expressed as the reduction in the number
of abdominal constrictions between the control animals and the
mice pre-treated with the compounds.
between placing the animal on the hot plate and the occurrence of
either licking of the hind paws, shaking the paw or jumping off the
surface was recorded as response latency. Mice with baseline
latencies of more than 20 s were eliminated from the study and the
cut-off time for the hot-plate latencies was set at 30 s. The animals
were treated 30 min before the assay. In order to correct for indi-
vidual differences in latencies, the antinociceptive data were con-
verted to percentage maximum possible effect (%MPE).
2.2.4. Formalin-induced nociception
The observation chamber was a glass cylinder of 20 cm in
diameter, with a mirror at a 45ꢀ angle to allow clear observation of
the animals’ paws. The mice were treated with 0.9% saline solution
(i.p.), or compound 3g (10, 30 and 60 mg kgꢁ1, i.p.), 30 min before
formalin injection. Each animal was placed in the chamber for
5 min before treatment, in order to acclimatize to the new envi-
ronment. The formalin test was carried out as described by Hun-
skaar and Tjolsen [18,19], with minor modifications. Twenty
microliters of a 2.5% formalin solution (0.92% formaldehyde) in 0.9%
saline solution were injected intraplantarly into the right hind
paw. The animal was then returned to the chamber, and the amount
of time spent licking the injected paw was considered as indicative
of pain. Two distinct phases of intensive licking activity were
identified: an early acute phase and a late or tonic phase (0e5 and
15e30 min after formalin injection, respectively).
2.2.8. Statistical analysis
The results are presented as mean ꢃ S.E.M., except for the ID
values (i.e. the dose of the compounds reducing the nociceptive
response by 50%, relative to the control value), which are reported
as geometric means, accompanied by their respective 95% confi-
dence limits. The ID50 value was determined by linear regression
from individual experiments using linear regression GraphPad
software (GraphPad software, San Diego, CA). The statistical
significance of the differences between the groups was detected by
ANOVA, followed by Dunnett’s multiple comparison tests. P-values
of less than 0.05 (P < 0.05) were considered indicative of
significance.
3. Results
2.2.5. Capsaicin-induced nociception
The procedure used was similar to that described previously
3.1. Chemistry
[20]. After the adaptation period, 20 ml of capsaicin (1.6 mg/paw)
was injected intraplantarly into the right hind paw. The animals
were observed individually for 5 min following capsaicin injection.
The amount of time spent licking the injected paw was timed
with a chronometer and was considered as indicative of noci-
ception. The animals were treated with the compound 3g via i.p.
(3, 6 and 10 mg kgꢁ1) 30 min prior to capsaicin injection, respec-
tively. The control animals received a similar volume of saline,
intraperitoneally.
The synthesis of the target compounds 3aeh were prepared in
only one step, in similar form to that previously studied by our
research group [14]. Generally, these conditions are used to obtain
chalcones, but when using the carboxybenzaldehyde as the starting
material and different acetophenones in the presence of alkali, the
expected chalcones are not obtained. In this reaction, the only
product is 3-phenacylphthalides, also known as benzofuranones,
which are produced in good yield. The spectral data of the
2.2.6. Glutamate-induced nociception
Table 1
The animals were treated with the compound 3g via i.p. (3, 10
Comparison of the antinociceptive activity of first generation benzofuranones and
reference drugs (AAS and PAR) in the acetic acid-induced pain model.
and 30 mg kgꢁ1) 30 min before glutamate injection, respectively. A
volume of 20 ml of glutamate solution (30 mmol/paw), was injected
O
O
O
intraplantarly under the surface of the right hind paw as described
previously [21]. After injection with glutamate, the animals were
individually placed in glass cylinders of 20 cm in diameter and
observed from 0 to 15 min. The time spent licking and biting the
injected paw was timed with a chronometer and considered
indicative of pain.
A
R
B
Substituents
Code
DI50
(
m
mol kgꢁ1
)
MI (%)
H
3a
3b
3c
3d
63.5 (44.5e90.6)
52.7 (47.2e65.7)
42.5 (36.0e13.3)
33.7 (29.2e39.0)
34.6 (26.2e45.8)
133.0 (73.0e243.0)
125.0 (104.0e250.0)
74.8
69.5
88.4
97.7
90.7
83.0
88.0
4-OCH3
4-CH3
4-Cl
2.2.7. Hot-plate test
The hot-plate test was used to measure response latencies,
according to the method described by Eddy and Leimback [22]. The
mice were treated with saline solution, morphine (10 mg kgꢁ1, s.c.)
or the compound 3g (10, 30 and 60 mg kgꢁ1, i.p.), and placed
individually on a hot plate maintained at 56 ꢃ 1 ꢀC. The time
3,4-Cl2
3e
AASa
PARa
a
F. de Campos-Buzzi et al [2].

C.J. Gonçalves et al. / European Journal of Medicinal Chemistry 56 (2012) 120e126
123
Table 2
4. Discussion
Comparison of the antinociceptive activity of second generation benzofuranones
and 3d from the first series in the acetic acid-induced pain model (10 mg kgꢁ1, i.p).
This study initially focused on the evaluation of a benzofuranone
series based on the Topliss manual method, using the acetic acid-
induced writing test. These compounds were obtained with
greater ease than previously reported. The literature data revealed
that 3-alkylphthalides are synthesized by reacting 2-bromo benz-
aldehyde with 1,3 dicarbonyl compounds under carbon monoxide
in the presence of a catalytic conditions of PdCl2(PPh3)2 [23].
Although one-pot synthesis was reported to obtained
3-phenacylphthalides, this reaction was performed in the presence
of trifluoroacetic acid [24]. The method used in our studies uses
more brand conditions than those reported in the literature, and
similar conditions used to synthesis of chalcones. For this reason,
we suggest that the chalcone intermediary is formed mechanisti-
O
O
O
A
R
B
Substituent (R)
Code
MI (%)
4-Cl
3d
3f
3g
3h
73.5
49.4
93.5
50.8
3,4-OCH3
3-OCH3
4-Br
synthesized compounds in the present investigation were in
accordance with their assigned structures.
cally. However, the rearrangement occurs between the a,b-unsat-
urated moiety and the carboxyl group to produce the
corresponding benzofuranones.
According to the ID50 values calculated, the potency ranking
for this first generation of active compounds was: 4-Cl > 3,4-
Cl2 > 3-CH3 > 4-OCH3 > H. Analyzing the influence of the elec-
tronic parameter of the substituent groups in relation to the inhi-
bition of constrictions caused by acetic acid, it was observed that
the compounds with electron-acceptor substituents were more
effective than the electron-donors or without substituents. All the
compounds were about 2e4-fold more active than the standard
drugs, such as acetylsalicylic acid and acetaminophen, used for
comparison.
3.2. Biological assays
In the writhing test, the first generation of compounds caused
69.5 at 97.7% of inhibition at 30 mg kgꢁ1 with dose-dependent
antinociceptive effect, with ID50 values ranging of 33.7 (29.2e39.0)
at 63.5 (44.5e90.6) m
mol kgꢁ1 (Table 1). Three more representa-
tive compounds were thus selected in accordance with the Topliss
method: 3,4-OCH3 (3f), 3-OCH3 (3g); 4-Br (3h) and were evaluated
in the same model at 10 mg kgꢁ1 (Table 2). These results were
determinants for selecting the substituent 3-OCH3 (3g), due to its
considerable improvement in the antinociceptive activity.
Besides the electronic parameter, the lipophilic parameter should
also be influencing the pharmacological activity, and on comparison
Compound 3g presented an ID50 value of 9.6 (7.8e11.9)
i.p. and an ID50 value of 257.1 (250.7e242.3)
m
mol kgꢁ1
m
mol kgꢁ1 by the oral
,
with the Topliss manual method, 2pe
p2 was determined as the
dominant parameter for selecting the second set of derivatives.
Three benzofuranones were therefore synthesized as repre-
sentative, from among those suggested by the Topliss method:
4-Br; 3-OCH3; 3,4-OCH3 and were evaluated in the writhing test at
10 mg kgꢁ1. Compound 3g (3-OCH3) was the most effective, causing
inhibition of 93.5%. Since it caused pronounced antinociceptive
action in this preliminary assay, it was then evaluated in more
detail in different classical models of pain in mice. Compound 3g
was evaluated as an antinociceptive agent, initially using the same
methodology i.e. the acetic acid-induced writhing test in mice,
administered intraperitoneally.
route (Fig. 2).
The formalin-test revealed that it inhibited the dose-dependent
manner the first phase of pain with a 38.3% of inhibition at
212.5
inhibition with a ID50 value of 174.3 (166.3e182.6)
m
mol kgꢁ1 and the second phase with a more significant
m
mol kgꢁ1
.
Formalin induced edema was also inhibited in a dose-dependent
manner (Fig. 3).
The administration of compound 3g produced dose-dependent
attenuation of capsaicin-induced neurogenic pain (Fig. 4), with ID50
value of 63.7 (59.2e68.5)
62.0%.
m
mol kgꢁ1, and maximal inhibition of
In comparison with the first generation of benzofuranones
classical according to Topliss (3aee) the ID50 were calculated in
order to evaluate the potency of this derivative which was about 3.5
times more active than compound 3d. This demonstrated that the
structural modification improved the activity, being about 14-fold
more active than the standard drugs, such as acetylsalicylic acid
and acetaminophen.
The results presented in Fig. 5 show that compound 3g, given
intraperitoneally, caused significant inhibition of glutamate-induced
nociception, with a mean ID50 value of 66.5 (58.3e75.8) mmol/kg and
maximal inhibition of 60.3%.
In the hot plate test, the animals did not show an increase in the
threshold of pain perception compared with the control group and
the pre-test carried out 24 h previously (Fig. 6).
Fig. 2. Effect of compound 3g (0.5, 1.0, 3.0 and 10 mg kgꢁ1), given intraperitoneally, (A) and (30, 60 and 100 mg kgꢁ1), given orally (B) against 0.6% acetic acid-induced abdominal
constrictions in mice. Each column represents the mean s.e.m. of six to eight experimental values. *p < 0.05; **p < 0.01.

124
C.J. Gonçalves et al. / European Journal of Medicinal Chemistry 56 (2012) 120e126
ID50 = 49.30 (47.04 - 51.66) mg.kg-1
A 180
B
174.27 (166.27 - 182.61) µmol.kg-1
250
160
140
120
100
80
60
40
20
14.2%
27.8%
**
13.7%
24.4%
200
150
100
50
38.3%
**
61.4%
**
0
0
Control
10 mg.kg-1
30mg.kg-1
60mg.kg-1
4.3%
Control
10 mg.kg-1
30mg.kg-1
60mg.kg-1
Compound 3g
Compound 3g
80
70
60
50
40
30
20
10
0
C
19.0%
39.40%
Control
10 mg.kg-1
30mg.kg-1
60mg.kg-1
Compound 3g
Fig. 3. Effect of compound 3g (10, 30 and 60 mg kgꢁ1), given intraperitoneally, against the first (0e5 min) phase (A) and against the second (15e30 min) phase (B) and edema (C) in
a formalin test in mice. Each column represents the mean s.e.m. of six to eight experimental values. *p < 0.05; **p < 0.01.
When evaluated by the oral route, compound 3g inhibited
abdominal constrictions by 79.1% at 100 mg/kg, being 2-times more
active than the standard drugs and demonstrating good oral
absorption, which is important for a drug candidate. Although the
writhing test is a non-specific model (i.e. anticholinergic and
antihistaminic and other agents also show activity in this test), the
fact that compound 3g significantly reduces the number of
abdominal constrictions induced by 0.6% acetic acid solution
suggests an antinociceptive potential, since this model is widely
used for analgesic screening and involves local peritoneal receptors
(cholinergic and histamine receptor) and the acetylcholine and
histamine mediators [25].
Compound 3g was also analyzed in the formalin-induced pain
test, a reported behavior model characterized by the first phase
(neurogenic), which is evoked by direct formalin stimulation of the
nerve endings followed by substance P release, and the second
phase, mainly due to a subsequent inflammation reaction in the
peripheral tissue [21,25,26].
The biphasic control of pain, which reflects different patholog-
ical processes, enables the elucidation of the possible mechanism
involved in analgesia [26]. Centrally acting drugs, such as opioids,
inhibit both phases of pain by equally involving the effect produced
by prostaglandins released at this level in response to inflammation
[26,27]. Some nonsteroidal anti-inflammatory drugs (NSAIDs) are
only peripheral acting drugs, such as acetylsalicylic acid, and
therefore reduce nociception only in the late phase, while another,
mefenamic acid, had a central site of action, as well as a peripheral
one, and affected both phases, although the second phase response
was inhibited by lower doses of these drugs than the first phase
response [27,28]. Compound 3g inhibited the first and second
phase of the formalin test, suggesting an involvement at both
central and peripheral levels, being much more active in the second
phase.
Compound 3g (10 mg/kg, i. p.) was also analyzed in the capsaicin
test, confirming the direct evidence of the antinociceptive effect of
this compound on neurogenic pain, since acute neurogenic
ID50 = 18.81 (16.50 - 21.45) mg/kg-1
250
200
150
100
50
66.49 (58.32 - 75.82) µmol/kg-1
47,3%
**
60,3%
**
0
Control
3mg.kg-1
10mg.kg-1
30mg.kg-1
Compound 3g
Fig. 4. Effect of compound 3g (3, 10 and 30 mg kgꢁ1) on licking/biting response
induced by intraplantarly injection of capsaicin in mice. Each group represents the
mean of six to eight experiments. **p < 0.01, compared with the corresponding control
value.
Fig. 5. Effect of compound 3g (3, 10 and 30 mg kgꢁ1) on licking/biting response
induced by intraplantarly injection of glutamate in mice. Each group represents the
mean of six to eight experiments. **p < 0.01, compared with the corresponding control
value.

C.J. Gonçalves et al. / European Journal of Medicinal Chemistry 56 (2012) 120e126
125
and is presented in Table 1 to assess the bioavailability of the
synthesized compounds. Initially we calculated the parameters
according to the Lipinski’s “rule of five”, which has been widely
used as a filter for substances that could likely be further developed
in drug design programs. This rule follows some principles: poor
absorption of permeation is more likely when there are more than
five H-bonds donors, the H-bond acceptors, the molecular weight
(MW) is greater than 500 and the calculated log P is greater than
five [31]. Molecules violating more than one of these rules may
have problems with bioavailability. According to Lipinski’ Rule the
synthesized compounds presented in this study do not show any
violation, these compounds being, theoretically, defined as
druglike.
The Human Intestinal Absorption (HIA) and penetration in
Blood-Brain Barrier (BBB) of the studied compounds were pre-
dicted by using a PreADMET [32] HIA is one of the most important
ADME properties. It is also one of the key steps during the drugs
transporting to their targets. Due to the diverse pathways of drugs
absorption, powerful descriptors related to carrier-mediated
transport and first-pass metabolism are needed for building
a useful prediction model for human oral bioavailability [33].
Topological polar surface area (TPSA), a surface belonging to
polar atoms, is a descriptor that was shown to correlate well with
passive molecular transport through membranes and, therefore,
allows prediction of transport properties of drugs in the intestines.
TPSA and volume are inversely proportional to %HIA [34].
Although HIA is considered as one of the important components
which influence the bioavailability, there are some differences in
the experimental values by compounds or their metabolisms. Yee
[35] consider three categories: Poorly absorbed (0e20%), moder-
ately absorbed (20e70%) and well absorbed (70e100%). All the
studied compounds predicted by PreADMET presented % HIA over
97%, being considered well absorbed.
The TPSA also is related with the bloodebrain barrier crossings.
Prediction BloodeBrain Barrier (BBB) penetration means predicting
whether compounds pass across the bloodebrain barrier. This is
crucial in pharmaceutical sphere because CNS-inactive compounds
do not should pass across it in order to avoid of CNS side effects.
Such evaluation also can present some differences in the experi-
mental values by compounds or their metabolisms. According to
Ma et al. [36], high absorption to CNS occur when BBB is more than
2.0; middle absorption is in the range 2.0e0.1 and low absorption is
less than 0.1.
Fig. 6. Effect of compound 3g (10, 30 and 60 mg kgꢁ1, i.p.) and morphine (5 mg kgꢁ1
s.c.) on the nociceptive effect in the hot plate test. Each column represents the mean
s.e.m. of six to eight experimental values. *p < 0.05; **p < 0.01.
,
inflammation can be evoked by intradermal injection of capsaicin.
Inflammation triggered by substances released from sensory
terminals is referred to as neurogenic inflammation. The
substances that contribute to neurogenic inflammation include
substance P, which results in plasma extravasation and calcitonin
gene-related peptide that triggers vasodilation [29]. These results
suggest a possible involvement of this compound with the antag-
onism of the vanilloid receptor.
Of great interest are the findings that demonstrate that the
antinociception caused by 3g could involve, at least in part, its
ability to interact with excitatory amino acids, as demonstrated by
the fact that it caused significant inhibition of hyperalgesia induced
by intraplantar injection of glutamate in mice. According to Beirith
and co-workers [21], intraplantar injection of glutamate into the
mouse hind paw results in rapid onset and short duration noci-
ception, the response of which is largely mediated by both
N-methyl-
D
-aspartate (NMDA) and non-NMDA
(a-amino-3-
hydroxyl-5-methyl-4-isoxazolepropionate (AMPA), kainite (KA)
and metabotropic) receptors and by the glycine modulatory site.
It is known that NSAIDs usually do not increase the pain
threshold in normal tissues in the way local anesthetics and
narcotics do. Likewise, the results obtained for compound 3g in the
hot plate test did not show any significant activity.
In recent years, estimate potential for drug have rapidly and
significantly increased in drug discovery. A computational study for
prediction of ADME properties of all molecules 3(aeh) was per-
formed in a web-based application denoted as Molinspiration [30]
With exception the compound 3a which exhibited a BB value
2.98, being the only compounds of series considered CNS-active, the
other compounds were classified as middle to low absorption to
Table 3
Pharmacokinetic properties important for ADME prediction of compounds 3aeh.
Code
MWa
Volume (A3)
TPSA (A2)b
NROTBc
HBAd
HBDe
log Pf
%HIAg
BBBh
Violations
3a
3b
3c
3d
3e
3f
252.27
282.29
266.30
286.71
321.16
312.32
282.29
331.16
225.19
250.74
241.75
238.73
252.26
276.28
250.74
243.08
43.38
52.61
43.38
43.38
43.38
61.84
52.61
43.38
3
4
3
3
3
5
4
3
3
4
3
3
3
5
4
3
0
0
0
0
0
0
0
0
2.96
3.01
3.41
3.64
4.24
2.61
2.99
3.77
97.91
98.07
97.82
97.61
97.65
98.50
98.07
97.61
2.98
0.18
0.04
0.18
0.15
0.05
0.02
0.18
0
0
0
0
0
0
0
0
3g
3h
a
MW, molecular weight.
b
c
TPSA, topological polar surface area.
NROTB, number of rotatable bonds.
HBA, number of hydrogen bond donors.
HBD, number of hydrogen bond acceptors.
log P, logarithm of compound partition coefficient between n-octanol and water.
%HIA, percentage of Human intestinal absorption.
d
e
f
g
h
BBB, bloodebrain barrier penetration.

126
C.J. Gonçalves et al. / European Journal of Medicinal Chemistry 56 (2012) 120e126
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Acknowledgments
We are grateful to Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES), Fundação de Apoio à Pesquisa
Científica e Tecnológica do Estado de Santa Catarina (FAPESC) and
Universidade do Vale do Itajaí (UNIVALI) for their financial support.
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