Antinociceptive effect of hydantoin 3-phenyl-5-(4-ethylphenyl)-imidazolidine-2,4-dione in mice

Article abstract of DOI:10.3390/molecules20010974

Imidazolidine derivatives, or hydantoins, are synthetic compounds with different therapeutic applications. Many imidazolidine derivatives have psychopharmacological properties, such as phenytoin, famous for its anticonvulsant efficacy, but also effective

Full text of DOI:10.3390/molecules20010974

Molecules 2015, 20, 974-986; doi:10.3390/molecules20010974
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antinociceptive Effect of Hydantoin 3-Phenyl-5-(4-ethylphenyl)-
imidazolidine-2,4-dione in Mice
Ronaldo Bezerra de Queiroz 1, Fabíola Lélis de Carvalho 1, Diogo Vilar da Fonsêca 1,
José Maria Barbosa-Filho 1, Paula Regina Rodrigues Salgado 1, Luciano Leite Paulo 1,
Ana Bárbara Maroja de Queiroz 1, Liana Clébia de Morais Pordeus 1, Severino Araújo de Souza 2,
Helivaldo Diogenes da Silva Souza 2, Bruno Freitas Lira 2 and Petrônio Filgueiras de Athayde-Filho 2,*
1
Center for Health Sciences, Federal University of Paraiba, João Pessoa PB 58059-900, Brazil;
E-Mails: qronaldo@ig.com.br (R.B.Q.); fabiolalelis@ltf.ufpb.br (F.L.C.);
divilar@hotmail.com (D.V.F.); jbarbosa@ltf.ufpb.br (J.M.B.-F.);
paulasalgado87@gmail.com (P.R.R.S.); luciano-sjp@hotmail.com (L.L.P.);
bmqueiroz1@gmail.com (A.B.M.Q.); liana@ccs.ufpb.br (L.C.M.P.)
2
Department of Chemistry, Federal University of Paraiba, João Pessoa PB 58059-900, Brazil;
E-Mails: sasindustrial@bol.com.br (S.A.S.); helivaldog3@gmail.com (H.D.S.S.);
brunofrlira@hotmail.com (B.F.L.)
* Author to whom correspondence should be addressed; E-Mail: athayde-filho@quimica.ufpb.br;
Tel.: +51-83-3216-7417.
Academic Editor: Derek J. McPhee
Received: 31 October 2014 / Accepted: 5 January 2015 / Published: 8 January 2015
Abstract: Imidazolidine derivatives, or hydantoins, are synthetic compounds with different
therapeutic applications. Many imidazolidine derivatives have psychopharmacological
properties, such as phenytoin, famous for its anticonvulsant efficacy, but also effective in
the treatment of neuropathic pain. The hydantoin, 3-phenyl-5-(4-ethylphenyl)-
imidazolidine-2,4-dione (IM-3), synthesized from the amino acid, glycine, was selected for
psychopharmacological studies in mice on the basis of its chemical and structural similarity
with phenytoin. The first step of this study was to define the LD50, which determined the
doses of 50, 100 and 200 mg/kg for subsequent tests. The results obtained from the
behavioral screening indicated that IM-3 produces decreased ambulation and analgesia in
mice. Motor coordination and anxiety behavior were not affected by treatment with IM-3,
as observed in the rotarod and elevated plus-maze tests, respectively. Regarding its
antinociceptive properties, IM-3 showed efficacy in the acetic acid-induced writhing test by

Molecules 2015, 20
increasing the latency of the first writhe and reducing the number of writhes, as well as
975
reducing the paw licking time in the second phase of the formalin test. The behavior of
treated animals exposed to the hot plate test, however, did not differ from that of the control
group. These data suggest that IM-3 has antinociceptive effects in mice, which is probably
mediated by anti-inflammatory mechanisms.
Keywords: hydantoin; psychopharmacology; antinociceptive; anti-inflammatory
1. Introduction
Amino acids are the building blocks of receptors, enzymes, peptide hormones and some
neurotransmitters and, thus, play important roles in the body. Even glycine, the simplest amino acid, is
essential to the normal functioning of the central nervous system (CNS), where it works as one of the
most important inhibitory neurotransmitters and is known to modulate pain perception [1,2].
Pain perception is related to the way in which the same noxious stimulus is experienced differently
when applied to different individuals [3]. It is determined not only by the amount of nociceptive input,
but mainly by descending modulatory mechanisms, which involve inhibitory and facilitatory pathways
from the CNS [4]. This process is influenced by the individual characteristics of each person and includes
age, ethnicity, gender, past experiences, memories and emotional state, among others [5–8].
Since glycine is directly involved in the pain modulation process, the importance of screening for
drugs obtained from this amino acid as potential tools in the management of pain is clear, especially
when considering the side effects of the therapeutic options currently available or even with their lack
of efficacy in some clinical conditions [9].
Several studies [10–12] have described the synthesis of amino acid compounds, their importance and
applications as intermediates for the synthesis of heterocyclics [13,14]. The present study aimed to
contribute to the pharmacological studies of imidazolidine compounds obtained from amino acids.
Accordingly, the hydantoins, heterocyclic structures obtained by organic synthesis from glycine and
characterized by the imidazolidine-2,4-dione ring, are considered potential drugs for the treatment of
pain. The attention focused on this class is also noted by the clinical efficacy of phenytoin, one of its
main representatives, as an ally in the treatment of neuropathic and cancer pain [15,16]. Despite its
extensive use, phenytoin has a narrow therapeutic index, which requires attention. Toxicological effects
include nystagmus, loss of smooth pursuit and eventually coma [17]. Given these disadvantages, the
development of a new improved drug is important to reduce such problems.
As other studies have demonstrated the efficacy of some hydantoin derivatives in animal pain
models [18,19], this study investigated the psychopharmacological activity of the hydantoin derivative,
3-phenyl-5-(4-ethylphenyl)-imidazolidine-2,4-dione (IM-3), in mice, particularly its antinociceptive activity.

Molecules 2015, 20
976
2. Results and Discussion
To obtain the target compound, the initial strategy was to synthesize C-4-ethylphenylglycine from
4-ethylbenzaldehyde, which reacts with sodium cyanide and ammonium chloride (Strecker synthesis),
followed by acid hydrolysis to form the IM-3 compound, according to our published procedure [20].
2.1. Acute Toxicity
IM-3 at doses of 400 and 800 mg/kg did not cause mortality in mice. In the groups treated with 1200
and 2000 mg/kg IM-3, 10% and 70% death was observed, respectively, while in the group that received
3000 mg/kg, there was 100% lethality. These results made it possible to calculate by nonlinear regression
the LD50 of IM-3 in mice by the intraperitoneal route, which was estimated to be 1358.9 mg/kg, with a
confidence interval of 1000–1584.9 mg/kg.
2.2. Behavioral Pharmacological Screening
At a dose of 50 mg/kg, increased grooming was observed after 240 min of observation, with no
change in the preceding minutes. At a dose of 100 mg/kg, no alteration was observed compared to the
control group, except an increase in grooming behavior at 120 min. Treatment with 200 mg/kg IM-3
resulted in analgesia in response to clamping the tail up to 180 min of observation. Also noticed was
reduced constipation and ambulation in the first 60 min and increased grooming behavior from 120 to
240 min of observation.
2.3. Rotarod Test
As shown in Table 1, treatment with 50, 100 and 200 mg/kg IM-3 did not affect the motor performance
of mice subjected to the rotarod test at 30, 60 or 120 min after administration.
Table 1. Effect of 3-phenyl-5-(4-ethylphenyl)-imidazolidine-2,4-dione (IM-3) on the rotarod
test in mice.
Time Spent on the Rotarod (Minutes)
Treatment
Dose (mg/Kg, i.p)
30
60
120
Control (10 mL/kg)
-
179.3 ± 0.7 167.8 ± 11.2 173.0 ± 6.0
136.4 ± 19.7 162.4 ± 15.5 161.4 ± 18.6
133.9 ± 18.9 159.4 ± 18.8 177.9 ± 1.4
146.9 ± 18.0 163.6 ± 15.2 179.4 ± 0.6
50
IM-3
100
200
Data are expressed as the mean ± SEM. (n = 8). ANOVA followed by Dunnett’s test.
2.4. Elevated Plus-Maze Test
None of the doses of IM-3 tested (50, 100 and 200 mg/kg) were able to change the number of entries
or the time spent in open arms (Table 2).

Molecules 2015, 20
977
Table 2. Effect of IM-3 on the elevated plus-maze test in mice.
Open Arms
Treatment
Dose (mg/kg, i.p.)
Number of Entries Duration (s)
Control (10 mL/kg)
IM-3
-
2.5 ± 0.8
3.2 ± 0.6
41.2 ± 14.3
41.5 ± 14.3
22.0 ± 9.5
50
100
0.5
2.2 ± 0.8
Diazepam
6.4 ± 0.6 **
90.7 ± 14.2 **
Data are expressed as the mean ± SEM. (n = 8). ANOVA followed by Dunnett’s test. ** p < 0.01.
2.5. Acetic Acid-Induced Writhing Test
As shown in Figure 1, only IM-3 at 200 mg/kg (630.7 ± 103.5 s) was able to significantly increase
the latency of the first contortion compared to the control group (213.3 ± 20.9 s). Figure 2 shows the
effects of IM-3 at doses of 50, 100 and 200 mg/kg i.p. on the number of writhes in mice. At doses
of 100 and 200 mg/kg, there was a significant decrease in the number of writhes (16.8 ± 1.7 and
6.0 ± 2.3 writhes, respectively) compared to the control group (29.6 ± 4.3 writhes).
Figure 1. Effect of IM-3 on the latency of the first contortion in the acetic acid-induced
writhing test in mice (n = 8). ANOVA followed by Dunnett’s test. *** p < 0.001.
Figure 2. Effect of IM-3 on the number of contortions in the acetic acid-induced writhing test
in mice (n = 8). ANOVA followed by Dunnett’s test. * p < 0.05, *** p < 0.001.

Molecules 2015, 20
978
2.6. Formalin Test
IM-3, at the doses tested (50 mg/kg: 91.6 ± 10.2 s; 100 mg/kg: 75.5 ± 5.0 s; 200 mg/kg: 78.0 ± 10.4 s),
showed no significant reduction in paw licking time in the first phase of the formalin test when compared
to the control group (93.0 ± 9.8 s) (Figure 3). According to the results presented in Figure 4, IM-3
decreased the paw licking time in the second phase of the formalin test at doses of 100 and 200 mg/kg
(58.5 ± 25.7 and 43.1 ± 24.2 s, respectively), when compared to the control group (160.1 ± 33.4 s).
Animals treated with morphine showed decreased paw licking time in both phases (16.3 ± 5.4 s in the
first phase and 20.6 ± 16.3 s in the second phase).
Figure 3. Effect of IM-3 on the first phase of the formalin test in mice (n = 8). ANOVA
followed by Dunnett’s test. *** p < 0.001.
Figure 4. Effect of IM-3 on the second phase of the formalin test in mice (n = 8). ANOVA
followed by Dunnett’s test. * p < 0.05; ** p < 0.01.
2.7. Hot Plate Test
Figure 5 shows the effects of IM-3 at doses of 50, 100 and 200 mg/kg i.p. on the time mice spent on
a hot plate. There was no indication of an antinociceptive effect of IM-3 at the doses tested with regard
to this parameter when compared to the control group.

Molecules 2015, 20
979
Figure 5. Effect of IM-3 on the hot plate test in mice (n = 8). ANOVA followed by Dunnett’s
test. * p < 0.05, *** p < 0.001.
2.8. Discussion
This study investigated the antinociceptive activity of 3-phenyl-5-(4-ethylphenyl)-imidazolidine-2,4-
dione (IM-3) in mice using behavioral methods.
The determination of LD50 was helpful to estimate the toxicity of IM-3 and to establish the nontoxic
doses that could be used, thus avoiding the possibility of toxic effects being confused with the true
psychopharmacological effects of IM-3. The LD50 of IM-3 by the i.p. route was 1358.9 mg/kg, with a
confidence interval of 1000–1584.9 mg/kg. Based on this result, doses of 50, 100 and 200 mg/kg were
chosen for the following behavioral experiments.
Behavioral psychopharmacological screening is a preliminary test of easy execution and able to detect
in a qualitative way some important central actions [21]. The doses of IM-3 tested caused decreased
ambulation and analgesia. These behavioral alterations are similar to those exhibited by substances that
lower CNS activity. The decrease in ambulation in mice may be the most common way of determining
if the test substance has depressor effects on the CNS, but it is imprudent to use this single parameter to
classify a drug. A reduction in the response to a mechanical stimulus is observed after treatment with
analgesic substances, and the presence of this behavior after administration of new drugs could be an
indication of antinociceptive activity [22].
Despite the depressant effect observed in behavioral screening, IM-3 did not impair the motor
coordination of mice, as observed in the rotarod test. Similar results have been reported for the hydantoin
derivatives, 5-phenylmethylenehydantoin (5-PMH), and its para-methoxy substituted derivative
(5-p-MPMH), while the ortho- and meta-substituted analogues (5-o-MPMH and 5-m-MPMH,
respectively) significantly reduce the ability of the mice to remain on the rotarod [23], demonstrating the
relevant role of the substituent groups on the effects of these drugs.
The performance of mice on the elevated plus-maze test was not affected by the treatment with IM-3,
indicating a lack of anxiolytic effect.
Luis et al. (2010) [20] synthesized a series of hydantoins and affirmed that, when given to mice at
doses of 250 and 500 mg/kg i.p., IM-3 reduced the number of writhes in the acetic acid-induced writhing
test, but did not alter the latency in the hot plate test. The current study complements and extends the
findings obtained by these authors on the antinociceptive activity of IM-3 using lower doses.

Molecules 2015, 20
980
The acetic acid-induced writhing test was chosen to confirm the antinociceptive effects of IM-3
evidenced at behavioral psychopharmacological screening. This test is a simple, fast and reliable model
to estimate the antinociceptive activity of substances. This test is sensitive to both central and peripheral
drugs [24,25], as intraperitoneal administration of acetic acid induces the release of prostaglandins and
CNS mediators [26,27].
IM-3 was found to reduce, in a dose-dependent manner, the number of abdominal contractions, as
demonstrated previously at higher doses [20], as well as increasing the latency of the first writhe at the
highest dose tested, when compared to the control group.
The formalin test is a chemical model of nociception that provides a greater specific response
compared to the acetic acid model [28]. It is considered a model of clinical pain [29], since two different
phases of nociception can be evaluated. The first phase begins immediately after formalin injection and
lasts for the first 5 min (neurogenic or acute pain), where it is related to direct chemical stimulation of
A δ fibers (Aδ-) and C-fiber nociceptors, and the release of excitatory amino acids, nitric oxide and P
substance. The second phase occurs between 15 and 30 min after formalin, and it is related to the release
of many pro-inflammatory mediators, such as bradykinin, prostaglandins and serotonin [30]. Drugs that
act at the central level, such as opioid analgesics, inhibit both phases of the formalin test; however, drugs
with peripheral action, such as anti-inflammatory ones, are effective only during the second phase.
None of the doses of IM-3 tested (50, 100 and 200 mg/kg) decreased the paw licking time during the
first phase of the formalin test, unlike morphine and other central acting drugs. Doses of 100 and 200 mg/kg
reduced paw licking time during the second phase, which is indicative of the anti-inflammatory activity
of IM-3. On the other hand, the hydantoins, 5-(1H-indol-3-yl-methylene)-2-thioxo-imidazolidin-4-one
and 3-(4-bromo-benzyl)-5-(1H-indol-3-yl-methylene)-2-thioxo-imidazolidin-4-one, studied by Guerra et al.
(2011) [18], reduced the paw licking time in both phases of the formalin test in mice.
To investigate the involvement of IM-3 in central structures, the hot plate test was performed, which
is a selective test that uses heat as a nociceptive stimulus to identify analgesic substances with a central
effect [31].
The doses of IM-3 tested (50, 100, 200 mg/kg) did not alter the latency of the thermal stimulus
response in the hot plate test, confirming the results obtained by Luis et al. (2010) [20]. Sudo et al.
(2010) [32], in turn, observed that the hydantoin derivative, PT-31 (3-(2-chloro-6-fluorobenzyl)-
imidazolidine-2,4-dione), probably exerts its antinociceptive response via central mechanisms, since it
increased the nociceptive threshold in mice subjected to the hot plate challenge after intraperitoneal
administration. The two indole-imidazolidine derivatives (LPSF/NN-52 and LPSF/NN-56) studied by
Guerra et al. (2011) [18] did not alter the latency response in the hot plate test in mice, just like IM-3.
These authors also demonstrated that the mechanism of action of both compounds is probably not
associated with a central action, but rather with their ability to reduce the levels of pro-inflammatory
cytokines, such as TNF-α and IL-1, and decrease leukocyte migration.

Molecules 2015, 20
981
3. Experimental Section
3.1. Animals
Swiss albino male mice (Mus musculus), aged 2 to 3 months old, weighing 25 to 35 g, were used.
The animals were housed in polyethylene cages and maintained under controlled temperature
(21 ± 1 °C), with a 12-hour light/dark cycle (lights on at 6:00 a.m.), with food (Purina® commercial
pellet feed, Paulínia-SP, Brazil) and water available ad libitum until 60 min prior to tests.
Mice were placed in polyethylene cages with 4 animals each at least 60 min before tests, to minimize
the eventual behavioral alterations of the animal. Each animal was used only once and euthanized at the
end of the test. All experimental procedures were analyzed and previously approved by the Ethics
Committee on Animal Use of Federal University of Paraiba, under Certificate No. 0404/10.
3.2. Drugs and Treatments
The synthetic compound, 3-phenyl-5-(4-ethylphenyl)-imidazolidine-2,4-dione (IM-3), shown in
Figure 6, was provided by the Laboratory of Research in Bioenergy and Organic Synthesis (LPBS) and
obtained as follows. Amino acid 4-ethylphenylglycine (2.0 g, 10 mmol) was dissolved with mixing in
35 mL of 10% sodium hydroxide. After dissolution, phenyl isocyanate (1.19 g, 10 mmol) was slowly
added with mixing. The solution was further mixed for 4 h and then acidified with HCl (12N) until a
precipitate formed. The precipitate was filtered and refluxed for 2 h in 100 mL of 6 N HCl. The
precipitate obtained was filtered and washed with ethanol and air dried. Recrystallization with distilled
water/ethanol (1:1) resulted in 2.87 g (90.0% yield) of white crystals (melting point: 216–218 °C).
1H-NMR (DMSO-d6, TMS) δ: 1.16 (t, 3H, CH3CH2 J = 7.6 Hz); 2.60 (q, 2H, CH3CH2 J = 7.6 Hz); 5.33
13
(s, 1H); 7.24–7.51 (m, 9H, aromatics); 8.97 (s, 1H, NH) ppm. C-NMR (DMSO-d6,TMS) δ: 15.7
(CH3CH2); 27.9 (CH3CH2); 59.8; 126.8; 127.1; 127.9; 128.2; 128.8; 132.2; 133.1; 144.3; 155.8; 171.9
(C=O) ppm (for the experimental details, see our published procedure [20]).
Figure 6. Chemical structure of hydantoin derivative IM-3 (3-phenyl-5-(4-ethylphenyl)-
imidazolidine-2,4-dione).
Minutes prior to its utilization, IM-3 was dissolved in 3.4% Tween 80 in distilled water. The control
group received the vehicle used for IM-3 solution (3.4% Tween 80). Morphine (Sigma, São Paulo-SP,
Brazil) was used as the standard drug in positive control groups and was diluted in distilled water. All
drugs and the vehicle were administered intraperitoneally (i.p.) in a volume of 0.1 mL/10 g of the
mouse’s weight.

Molecules 2015, 20
982
3.3. Acute Toxicity
Acute toxicity was estimated by calculating the 50% lethal dose (LD50) in mice by the intraperitoneal
(i.p.) route. Animals were treated with IM-3 at doses of 400, 800, 1200, 2000 and 3000 mg/kg and were
observed every 24 h for a period of 72 h to check for any deaths and to determine the LD50 of IM-3 [33].
3.4. Behavioral Pharmacological Screening
The experimental protocol established by Almeida et al. [21] was employed to evaluate the effect of
IM-3 on the central nervous system. In this preliminary method, mice received IM-3 at doses of 50, 100
and 200 mg/kg, and resultant behavioral alterations in comparison with the control group (which received
only the vehicle) were recorded at 30, 60, 120 and 240 min after treatment.
3.5. Motor Incoordination (Rotarod Test)
The rotarod test measures the effect of muscle relaxation and motor incoordination produced by drugs.
It consists of placing mice on a bar that rotates at a constant speed and determining their ability to remain
on it [34].
Animals were pre-selected 24 h prior to the experiment, according to their ability to remain on the
rotating bar (7 rpm) for at least 3 min in 3 attempts. On the day of the experiment, the pre-selected mice
were divided into groups (n = 8), treated with vehicle or IM-3 (50, 100 or 200 mg/kg), and evaluated at
30, 60 and 120 min after treatment. The parameter evaluated was the time spent on the bar for a period
of up to 3 min. Animals that fell could be put back on the bar for up to 3 attempts.
3.6. Anxiolytic Activity (Elevated Plus-Maze Test)
The elevated plus-maze test is a simple and useful assay to access anxiety-related behavior induced
by pharmacological agents in rodents. It consists of an elevated maze with two open and two enclosed
arms and is based on the aversion of rats and mice to high and open environments, which is reduced by
treatment with anxiolytic drugs [35].
Mice were divided into five groups (n = 8) and treated with the vehicle, IM-3 (50, 100 or 200 mg/kg)
or 0.5 mg/kg diazepam (standard drug). Thirty minutes after administration, animals were individually
placed in the center of the elevated plus-maze, with the nose facing one of the open arms, and observed
for 5 min to record the number of entries and the time spent in the open arms.
3.7. Antinociceptive Activity Evaluation of IM-3
3.7.1. Acetic Acid-Induced Writhing Test
The acetic acid-induced writhing test is based on the irritation evoked by intraperitoneal injection of
0.85% acetic acid in mice, which involves the stimulation of nociceptors and consequent behavioral
reactions. The nociceptive effect is characterized by abdominal contortions followed by hind leg extension.
In general, drugs with analgesic properties reduce or even inhibit this behavior [36].
Mice were divided into five groups (n = 8) and treated with vehicle, IM-3 (50, 100 or 200 mg/kg) or
6 mg/kg morphine. Thirty minutes later, each animal received 0.85% acetic acid and was placed in

Molecules 2015, 20
983
individual polyethylene boxes to record the latency to the first contortion and the number of writhes
from 5 to 15 min after acetic acid injection
3.7.2. Formalin Test
Mice were divided into five groups (n = 8) and treated with the vehicle, IM-3 (50, 100 or 200 mg/kg)
or 10 mg/kg morphine (standard drug). After thirty minutes, 20 μL of 2.5% formalin were injected into
the intraplantar region of the right hind paw of the mice. Afterwards, these animals were individually
put in observation boxes, and the paw licking time was recorded for five minutes (first phase). After
10 min, the same parameter was determined for 15 min (second phase) [30,37].
3.7.3. Hot Plate Test
This test consists of quantifying the reaction time of the animal to thermal stimulus, to evaluate
analgesic activity mediated by central mechanisms [38,39].
Animals were subjected to a previous selection (without drug administration) and were considered
suitable if they responded (tried to jump or lick the hind paws) for up to 15 s after being put on the hot
plate (basal reading), which was set at 52 °C.
Mice were divided into five groups (n = 8) and treated with vehicle, IM-3 (50, 100 or 200 mg/kg)
or 10 mg/kg morphine (standard drug). Thirty minutes after treatment, the animals were individually put
on the hot plate, and the latency of response was recorded as the indication of antinociceptive action.
This procedure was repeated at 60 and 120 min after administration. The animals stayed on the plate for
a maximum of 30 s, to avoid tissue damage [22].
3.8. Statistical Analysis
The results presented were obtained using ANOVA, followed by Dunnett’s test. LD50 was calculated
by non-linear regression. Data were analyzed using the GraphPad Prism program version 4.02, and
the values obtained, except LD50, were expressed as the mean ± standard error of mean. p < 0.05 was
considered significant.
4. Conclusions
Taken together, the results obtained in this work strongly suggest that the synthetic compound,
IM-3, at the doses tested and by the intraperitoneal route, stimulates behavioral alterations in mice like
CNS depressant drugs and reduces nociception, possibly through anti-inflammatory pathways. Further
studies with appropriate pharmacological tools are necessary to elucidate the mechanisms involved.
Acknowledgments
The authors would like to express their sincere thanks to J.C. Duarte for his technical assistance.
This work was supported by the Brazilian agencies, CNPq and CAPES. A. Leyva helped with English
editing of the manuscript.

Molecules 2015, 20
984
Author Contributions
R.B.Q. and J.M.B.-F. designed the research study; P.R.R.S., L.L.P., D.V.F. and A.B.M.Q. performed
the experiments (pharmacological experiments); S.A.S., H.D.S.S., B.F.L. and P.F.A.-F. performed the
experiments (chemical isolation); L.C.M.P. analyzed the data; F.L.C. wrote the paper. All authors
discussed the results and commented on the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Dohi, T.; Morita, K.; Kitayama, T.; Motoyama, N.; Morioka, N. Glycine transporter inhibitors as a
novel drug discovery strategy for neuropathic pain. Pharmacol. Ther. 2009, 123, 54–79.
2. Miraucourt, L.S.; Dallel, R.; Voisin, D.L. Glycine inhibitory dysfunction turns touch into pain through
PKCgamma interneurons. PLoS One 2007, 2, e1116.
3. Ossipov, M.H.; Dussor, G.O.; Porreca, F. Central modulation of pain. J. Clin. Investig. 2010, 120,
3779–3787.
4. Rhudy, J.L.; Bartley, E.J.; Palit, S.; Kerr, K.L.; Kuhn, B.L.; Martin, S.L.; DelVentura, J.L.; Terry, E.L.
Do sex hormones influence emotional modulation of pain and nociception in healthy women?
Biol. Psychol. 2013, 94, 534–544.
5. Riley, J.L.; Cruz-Almeida, Y.; Glover, T.L.; King, C.D.; Goodin, B.R.; Sibille, K.T.; Bartley, E.J.;
Herbert, M.S.; Sotolongo, A.; Fessler, B.J.; et al. Age and race effects on pain sensitivity and
modulation among middle-aged and older adults. J. Pain 2014, 15, 272–282.
6. Craft, R.M. Modulation of pain by estrogens. Pain 2007, 132, S3–S12.
7. Horsham, S.; Chung, M.C. Investigation of the relationship between trauma and pain catastrophising:
The roles of emotional processing and altered self-capacity. Psychiatry Res. 2013, 208, 274–284.
8. Coghill, R.C. Individual differences in the subjective experience of pain: New insights into
mechanisms and models. Headache 2010, 50, 1531–1535.
9. Ossipov, M.H.; Porreca, F. Challenges in the development of novel treatment strategies for
neuropathic pain. NeuroRx 2005, 2, 650–661.
10. Lira, B.F.; Athayde-Filho, P.F.; Miller, J.; Simas, A.M.; Dias, A.F.; Vieira, M.J. Synthesis and
characterization of some new mesoionic 1,3-thiazolium-5-thiolates via cyclodehydration and in situ
1,3-dipolar cycloaddition/cycloreversion. Molecules 2002, 7, 791–800.
11. De Athayde-Filho, P.F.; Miller, J.; Simas, A.M.; Lira, B.F.; de Souza Luis, J.A.;
Zuckerman-Schpector, J. Synthesis, characterization and crystallographic studies of three
2-aryl-3-methyl-4-aryl-1,3-thiazolium-5-thiolates. Synthesis (Stuttgart) 2003, 5, 685–690.
12. Lira, B.F.; Miller, J.; Simas, A.M.; Athayde-Filho, P.F.; Dias, A.F.; Silva, R.O.; Oliveira, V.C.
Synthesis and complete assignments of 1H and 13C NMR spectra of mesoionic
2-(ptrifluoromethylphenyl)-3-methyl-4-(p-tolyl)-1,3-thiazolium-5-thiolate and 2-(p-chlorophenyl)-
3-methyl–4-(p-isopropylphenyl)-1,3-thiazolium-5-thiolate. ARKIVOC 2004, 6, 12–21.

Molecules 2015, 20
985
13. Bosco, C.A.C.; Maciel, G.S.; Rakov, N.; de Araújo, C.B.; Acioli, L.H.; Simas, A.M.;
Athayde-Filho, P.F.; Miller, J. Probing the nuclear susceptibility of mesoionic compounds using
two-beam coupling with chirp-controlled pulses. Chem. Phys. Lett. 2007, 449, 101–106.
14. Pilla, V.; de Araújo, C.B.; Lira, B.F.; Simas, A.M.; Miller, J.; Athayde-Filho, P.F. Nonlinear
absorption of new mesoionic compounds. Opt. Commun. 2006, 264, 225–228.
15. Mantegazza, M.; Curia, G.; Biagini, G.; Ragsdale, D.S.; Avoli, M. Voltage-gated sodium channels
as therapeutic targets in epilepsy and other neurological disorders. Lancet Neurol. 2010, 9, 413–424.
16. Yajnik, S.; Singh, G.P.; Singh, G.; Kumar, M. Phenytoin as a co-analgesic in cancer pain. J. Pain
Symptom Manag. 1992, 7, 209–213.
17. Imam, S.H.; Landry, K.; Kaul, V.; Gambhir, H.; John, D.; Kloss, B. Free phenytoin toxicity. Am. J.
Emerg. Med. 2014, 32, 1301.e3–1301.e4.
18. Guerra, A.S.; Malta, D.J.; Laranjeira, L.P.; Maia, M.B.; Colaço, N.C.; Lima, M.C.; Galdino, S.L.;
Pitta, I.R.; Gonçalves-Silva, T. Anti-inflammatory and antinociceptive activities of indole-imidazolidine
derivatives. Int. Immunopharmacol. 2011, 11, 1816–1822.
19. Gerona-Navarro, G.; González-Muñiz, R.; Fernández-Carvajal, A.; González-Ros, J.M.;
Ferrer-Montiel, A.; Carreño, C.; Albericio, F.; Royo, M. Solid-phase synthesis of a library of
amphiphatic hydantoins. Discovery of new hits for TRPV1 blockade. ACS Comb. Sci. 2011, 13,
458–465.
20. Luis, J.A.S.; Barbosa-Filho, J.M.; Lira, B.F.; Medeiros, I.A.; de Morais, L.C.S.M.L.;
dos Santos, A.F.; de Oliveira, C.S.; Athayde-Filho, P.F. Synthesis of new imidazolidin-2,4-dione
and 2-thioxoimidazolidin- 4-ones via C-phenylglycine derivatives. Molecules 2010, 15, 128–137.
21. Almeida, R.N.; Falcão, A.C.G.M.; Diniz, R.S.T.; Quintans-Júnior, L.J.; Polari, R.M.;
Barbosa-Filho, J.M.; Agra, M.F.; Duarte, J.C.; Ferreira, C.D.; Antoniolli, A.R.; et al. Methodology
for evaluation plants with activity in the central nervous system and some experimental data.
Rev. Bras. Farm. 1999, 80, 72–76.
22. Almeida, F.R.C.; Oliveira, F.S. Avaliação de drogas analgésicas de ação central. In Psicofarmacologia:
Fundamentos Práticos; Almeida, R.N., Ed.; Guanabara Koogan: Rio de Janeiro, Brazil, 2006;
pp. 179–188.
23. Wong, P.T.; Tan, S.F. Pharmacological profiles of 5-phenylmethylenehydantoin and its methoxy
substituted derivatives. Jpn. J. Pharmacol. 1989, 49, 309–315.
24. Le Bars, D.; Gozariu, M.; Cadden, S.W. Animal models of nociception. Pharmacol. Rev. 2001, 53,
597–652.
25. Vogel, H.G.; Vogel, W.H. Drug Discovery and Evaluation, Pharmacological Assays; Springer:
Berlin, Germmany, 1997; pp. 370–371.
26. Deraedt, R.; Jouquey, S.; Delevalle, F.; Flahaut, M. Release of prostaglandins E and F in
an algogenic reaction and its inhibition. Eur. J. Pharmacol. 1980, 61, 17–24.
27. Duarte, I.D.G.; Nakamura, M.; Ferreira, S.H. Participation of the sympathetic system in acetic
acid-induced writhing in mice. Braz. J. Med. Biol. Res. 1988, 21, 341–343.
28. Shibata, M.; Ohbubo, T.; Takahashi, H.; Inoki, R. Modified formalin test: Characteristic biphasic
pain response. Pain 1989, 38, 347–352.

Molecules 2015, 20
986
29. Tjölsen, A.; Hole, K. Animals models of analgesia. In The Pharmacology of Pain; Handbook of
Experimental Pharmacology Series, v. 25; Dickenson, A.H., Besson, J.M., Eds.; Springer-Verlag:
Berlin, Germany, 1997; pp. 1–20.
30. Hunskaar, S.; Hole, K. The formalin test in mice: Dissociation between inflammatory and
non-inflammatory pain. Pain 1987, 30, 103–104.
31. Woolfe, H.G.; MacDonald, A.D. The evaluation of the analgesic action of pethidine hydrochloride.
J. Pharmacol. Exp. Ther. 1944, 80, 300–307.
32. Sudo, R.T.; Calasans-Maia, J.A.; Galdino, S.L.; Lima, M.C.; Zapata-Sudo, G.; Hernandes, M.Z.;
Pitta, I.R. Interaction of morphine with a new alpha2-adrenoceptor agonist in mice. J. Pain 2010, 11,
71–78.
33. Litchfield, J.T.; Wilcoxon, F.A. Simplified method of evaluating dose-effect experiments.
J. Pharmacol. Exp. Ther. 1949, 96, 99–113.
34. Dunham, N.W.; Miya, T.S. A note on a simple apparatus for detecting neurological deficit in rats
and mice. J. Am. Pharm. Assoc. 1957, 46, 207–208.
35. Walf, A.A.; Frye, C.A. The use of the elevated plus maze as an assay of anxiety-related behavior in
rodents. Nat. Protoc. 2007, 2, 322–328.
36. Collier, H.O.J.; Dinneen, L.C.; Johnson, C.A.; Schneider, C. The abdominal constriction response
and its suppression by analgesic drugs in mouse. Br. J. Pharmacol. 1968, 32, 295–310.
37. Souza, M.M.; Madeira, A.; Berti, C.; Krogh, R.; Yunes, R.A.; Cechinel-Filho, V. Antinociceptive
properties of the methanolic extract obtained from Ipomoea pes-caprae (L.) R. Br. J. Ethnopharmacol.
2000, 69, 85–90.
38. Silva, M.G.; Oliveira, F.S.; Quintans-Júnior, L.J.; Oliveira, T.M.L.; Diniz, M.F.F.M. Investigação
do efeito analgésico central e anti-inflamatório de Conocliniopsis prasiifolia (DC) R.M. King & H.
Robinson em Roedores. Acta Farm. Bonaer. 2005, 24, 533–537.
39. Al-ghamdi, M.S. The anti-inflammatory, analgesic and antipyretic activity of Nigella sativa.
J. Ethnopharmacol. 2001, 76, 45–48.
Sample availability: Samples of the compound hydantoin 3-phenyl-5-(4-ethylphenyl)-imidazolidine-
2,4-dione are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).