Synthesis of new 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea and urea derivatives with anti-nociceptive activity

Article abstract of DOI:10.1016/j.bmc.2008.08.019

Chalcones or 1,3-diaryl-2-propen-1-ones are known to be useful for treating pain, inflammation, and certain diseases although their uses have not been scientifically verified. Due to the limitations of opioid and NSAID therapy, there is a continuing search for new analgesics. A series of novel new 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea and urea derivatives were synthesized and evaluated against writhing test in mice, following the aromatic substitution pattern proposed by Topliss. The results of the preliminary bioassays indicate that compound 3 presents promising anti-nociceptive activity in acetic acid-, formalin-, and glutamate-induced pain in mice, compared with some well-known non-steroidal anti-inflammatory and analgesic drugs.

Full text of DOI:10.1016/j.bmc.2008.08.019

Bioorganic & Medicinal Chemistry 16 (2008) 8526–8534
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of new 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea
and urea derivatives with anti-nociceptive activity
b
b,
b
Lorena dos Santos a, , Luíse Azevedo Lima , Valdir Cechinel-Filho , Rogério Corrêa ,
*
*
Fátima de Campos Buzzi b, Ricardo José Nunes a,
*
a Departamento de Química, Curso de Pós-Graduação em Química, Universidade Federal de Santa Catarina/UFSC, Florianópolis, Santa Catarina, Brazil
b Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale de Itajaí/UNIVALI, Itajaí, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chalcones or 1,3-diaryl-2-propen-1-ones are known to be useful for treating pain, inflammation, and cer-
tain diseases although their uses have not been scientifically verified. Due to the limitations of opioid and
NSAID therapy, there is a continuing search for new analgesics. A series of novel new 1-phenyl-3-{4-
[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea and urea derivatives were synthesized and evaluated
against writhing test in mice, following the aromatic substitution pattern proposed by Topliss. The results
of the preliminary bioassays indicate that compound 3 presents promising anti-nociceptive activity in
acetic acid-, formalin-, and glutamate-induced pain in mice, compared with some well-known non-ste-
roidal anti-inflammatory and analgesic drugs.
Received 23 June 2008
Revised 29 July 2008
Accepted 4 August 2008
Available online 8 August 2008
Keywords:
Chalcones
Thioureas
Ureas
Ó 2008 Elsevier Ltd. All rights reserved.
Anti-nociception
1. Introduction
anti-nociceptive activity against different models of pain tested
in mice. The most active compound was evaluated in more detail
Chalcones have been considered the main biological precursors
for the biosynthesis of flavonoids, which are common components
of the human diet. Recent studies on the biological evaluation of
chalcones have revealed many compounds, with a wide range of
biological effects, including anti-cancer,1 anti-inflammatory,2,3
anti-mitotic,4 anti-microbial,5 anti-leishmanial,6,7 anti-malarial,8,9
anti-tubercular,10 cardiovascular,11 anti-lipidemic,12 and anti-
hyperglycemic agents.13 Previous studies carried out in our
laboratories have demonstrated that some chalcones,14 or those
compounds derived from xanthoxyline, have promising analgesic
action in mice.15–17
Reactions of chalcones with isocyanates or isothiocyanates and
amines give ureas and thioureas which are of considerable indus-
trial importance, and are linked to a series of biological activities
including herbicidal activity,18 inhibition of nitric oxide,19 anti-
microbial,20 anti-HIV,21 anti-viral,22 HDL-elevating,11 and analgesic
properties.23,24 Some 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]-
phenyl}-urea derivatives exhibited anti-inflammatory3,25 and
anti-malarial activity.8,9 In view of the need to discover new anal-
gesic agents, and our previous positive results on chalcones, we
have synthesized new 1-phenyl-3-{4-[(2E)-3-phenylprop-2-en-
oyl]phenyl}-thiourea and urea derivatives, and evaluated the
and compared with some reference drugs.
2. Results and discussion
2.1. Synthesis and chemical characterization
In order to determine the possible anti-nociceptive action
of
1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea
and urea derivatives, we initially prepared: 1-(4-acetylphenyl)-3-
(4-chlorophenyl)thiourea 1 and 1-(4-acetylphenyl)-3-(4-chloro-
phenyl)urea 2, which are obtained according to Scheme 1. A
procedure based on
a Claisen–Schmidt condensation was
developed for the syntheses of five 1-phenyl-3-{4-[(2E)-3-phenyl-
prop-2-enoyl]phenyl}-ureas and five new 1-phenyl-3-{4-[(2E)-3-
phenylprop-2-enoyl]phenyl}-thioureas (Scheme 1) following the
aromatic substitution pattern proposed by Topliss.26,27 The yields
and melting points of the compounds 3–12 are listed in Table 1.
The chemical identification data (1H NMR, 13C NMR, IR, and ele-
mental analysis) confirmed the structures of compounds 3–12. In
the infrared spectra of compounds 3–12, it was possible to observe
the absorptions between 3296 and 3344 cmꢀ1 relating to NH
stretch, absorptions in 1638–1660 cmꢀ1 from
a,b-unsaturated car-
bonyl moiety stretch and absorptions in 1708–1713 cmꢀ1 from
urea carbonyl moiety stretching (Table 1). Thiocarbonyl is less
polar than the carbonyl group, and the link C@S is considerably
* Corresponding authors. Tel.: +55 21 37216844; fax: +55 21 33417501.
E-mail addresses: lorennasantos@gmail.com (L. dos Santos), cechinel@univali.br
(V. Cechinel-Filho), nunes@qmc.ufsc.br (R.J. Nunes).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.019

L. Santos et al. / Bioorg. Med. Chem. 16 (2008) 8526–8534
8527
O
NCX
NH
NH
CH3
Me2CO
X
O
+
Cl
room temperature
H2N
X= S
(1)
CH3
Cl
X= O (2)
H
O
MeOH
NaOH
Y
NH
NH
X
Cl
Y
(3)
X=S; Y= H
O
X=S; Y= Cl-4 (4)
(5)
X=S; Y= Cl2-3,4
X=S; Y= OMe-4 (6)
X=S; Y= Me-4 (7)
X=O; Y= H (8)
(9)
X=O; Y= Cl-4
X=O; Y= Cl2-3,4
X=O; Y= OMe-4 (11)
(12)
(10)
X=O; Y= Me-4
Scheme 1.
weaker, consequently, the band is not intense and it is located at
lower frequencies than carbonyl, and is therefore more susceptible
to the effects of coupling, their identification often being difficult
and uncertain.28
The 1H NMR spectra for all the synthesized compounds show
signals at 9.5 ppm relating to hydrogens attached to the nitrogen.
The signals for aromatic hydrogens are between 7.0 and 8.3 ppm.
In this same region are the vinylic protons of trans olefinic protons
which have larger coupling constants than those of their cis iso-
mers. All the structures were geometrically pure and E configured
Table 1
(JHa
–Hb = 14–17 Hz). Some studies on chalcone derivatives29,30
Structure and physical data of 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-
thiourea and urea derivatives
have shown this configuration exclusively.
Through the 13C NMR data, a sign can be seen at 187 ppm,
relating to chalcone carbonyl. This is followed by the sign for
thiourea carbonyl, at 179 ppm, and the sign for urea carbonyl
at 152 ppm. The quaternary and tertiary carbons are distributed
in the 115–141 ppm region, as well as can be observed, in this
band of chemical displacement, signals relating to olefinic
carbons.
6"
3"
NH
NH
5
2
6'
3'
5"
4"
1"
2"
6
1
4
3
1'
2'
5'
4'
X
Cl
Y
O
Compound
X
Y
Mpa (°C)
Yield (%)
2.2. Evaluation of anti-nociceptive activity
Found
Lit.
3
4
5
6
7
8
9
10
11
12
S
S
S
S
–H
4-Cl
3,4-Cl2
4-OCH3
4-CH3
–H
178–180
187–190
172–174
195–197
208–210
202–204
249–251
198–200
208–210
217–219
Unknown
Unknown
Unknown
Unknown
Unknown
223–224b
260–264b
Unknown
218–219b
232–233b
50
90
80
70
37
76
87
87
88
93
This work aims to identify the effectiveness of 1-phenyl-3-{4-
[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea and urea deriva-
tives in a number of animal models of pain. In order to select
the more active compounds, all the synthesized compounds were
evaluated using the writhing test in mice, as a preliminary trial.
A significant proportion of the work will be carried out using
cells and tissues isolated from humans or from humanely killed
animals, that is, in vitro. However, pain is a highly complex pro-
cess requiring an input from many parts of the nervous system
throughout the body, and, for this reason, in vitro testing alone
S
O
O
O
O
O
4-Cl
3,4-Cl2
4-OCH3
4-CH3
a
Melting points were uncorrected.
Dominguez et al., 2005.9
b

8528
L. Santos et al. / Bioorg. Med. Chem. 16 (2008) 8526–8534
Table 2
is not enough to determine if new medicines will be effective
analgesics.31,32
Comparison of the anti-nociceptive effects of 1-phenyl-3-{4-[(2E)-3-phenylprop-2-
enoyl]phenyl}-thiourea and urea derivatives with non-steroidal analgesic drugs (ASA,
dipyrone, and acetaminophen) given intraperitoneally in mice, in the writhing test
In a preliminary trial, as shown in Figure 1, all the compounds
tested with the exception of compound 10 significantly inhibited
acetic acid-induced abdominal constrictions when administered
intraperitoneally at 10 mg/kg, compared with dipyrone, acetylsali-
cylic acid, and acetaminophen, which were used as reference
drugs.
Due to the excellent results obtained in this test (Fig. 1), all the
compounds were evaluated in more detail in the same model. The
results shown in Table 2 indicate that nine of the compounds eval-
uated, five 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-
thioureas (3–7) and four 1-phenyl-3-{4-[(2E)-3-phenylprop-2-en-
oyl]phenyl}-ureas (8, 9, 11, and 12), which were tested in the
writhing test, caused dose-dependent anti-nociceptive effects
when given by the intraperitoneal route, inhibiting acetic acid-in-
duced writhing responses in mice, with the exception of compound
10, which was inactive at the dose of 60 mg/kg. 1-phenyl-3-{4-
[(2E)-3-phenylprop-2-enoyl]phenyl}-thioureas presented a calcu-
6"
NH
NH
5
6'
5"
4"
1"
2"
6
1
4
3
1'
2'
5'
4'
X
3'
3"
2
Cl
Y
O
Compound
X
Y
ID50
(
l
mol/kg, ip)a
MIb
94.5 1.1**
3
S
–H
3.03 (1.99–4.58)
4
5
6
7
8
9
10
11
12
S
S
S
S
O
O
O
O
O
4-Cl
3,4-Cl2
4-OCH3
4-CH3
–H
7.59 (6.26–9.25)
6.15 (4.91–7.69)
100.0 0.0**
97.5 0.6**
92.0 1.4**
75.3 4.5**
74.6 4.1**
51.0 2.9**
21.6 3.8**
84.4 2.0**
42.8 4.2**
10.37 (8.21–13.07)
13.14 (11.0–15.72)
7.25 (6.11–8.63)
45.38 (37.71–54.63)
Not dose-dependent
31.05 (11.60–14.85)
39.70 (35.12–44.87)
4-Cl
3,4-Cl2
4-OCH3
4-CH3
lated ID50 value (95% confidence limit) of 3.03–13.14 lmol/kg,
ASA
Dip.
ACE
—
—
—
—
—
—
133.0 (73.0–243.0)
162.0 (88.0–296.0)
125.0 (104.0–150.0)
35.0 2.0**
33.0 3.5**
38.0 1.0**
with maximum inhibition of 100 %, while the 1-phenyl-3-{4-
[(2E)-3-phenylprop-2-enoyl]phenyl}-ureas were less active with
ID50 values ranging from 7.25 to 45.38 lmol/kg; with maximum
inhibition of 84.4%. Some of the compounds tested were several
times more active than the analgesic drugs used as reference: ace-
tic salicylic acid (ASA), dipyrone, and acetaminophen (ACE), which
a
95% confidence limit. Each group represents the mean of six to eight animals.
Compounds, acetyl salicylic (ASA), dipyrone (Dip.) and acetaminophen (ACE) were
given intraperitoneally at 10 mg/kg.
b
Maximal inhibition.
p < 0.01 compared with the corresponding control value.
presented ID50 values of 138.7 (73–243)
mol/kg, and 125.0 (104–150) mol/kg, respectively, in the same
experimental model.
lmol/kg, 162.2 (88–296)
**
l
l
The results reported in the present investigation demonstrated
for the first time that 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]-
phenyl}-thioureas exert anti-nociceptive effects in mice. It is possi-
ble to observe in Table 2 that the percentage of maximal inhibition
of 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thioureas
showed higher values than that of 1-phenyl-3-{4-[(2E)-3-phenyl-
prop-2-enoyl]phenyl}-ureas, besides the lowest values of ID50. In
both series, the compounds without substituent in the aromatic
ring (from aldehyde) were more active. Of these, compound 3 pro-
duced the most potent and dose-dependent action in writhing test
model in mice, being approximately 47 times more active than the
reference drugs in the intraperitoneal treatment.
70
60
50
40
30
20
10
0
ID50 = 377.5 (344.63 - 413.48) mol/kg
23.5%
*
43.4%
**
55.0%
**
Control
50
100
200
Administered orally, compound 3 presents maximal inhibition
Compound 3 (mg/kg, p.o.)
of 55.0% at
(344.63–413.48)
tent than the standard drugs (ASA and ACE) used as reference,
which presented ID50 values of 605 (516–705) mol/kg and
a
dose of 200 mg/kg with ID50 value of 377.5
lmol/kg (Fig. 2), being about two times more po-
Figure 2. Effect of compound 1-(4-chlorophenyl)-3-{4-[(2E)-3-phenylprop-2-
enoyl]phenyl}thiourea 3, administered orally, against acetic acid-induced abdom-
inal constrictions in mice. Each column represents the mean s.e.m. of six
experimental values. **p < 0.01.
l
ID50 = 1145 (708–1846)
lmol/kg, respectively, in the same experi-
60
50
40
30
21.6%
**
33%
**
42.8%
**
35% 38%
** **
51%
**
75.3% 74.6%
** **
20
10
0
84.4%
**
94.5%
**
92%
**
97.5%
**
100%
**
4
C
3
5
6
7
8
9
10
11
12
Dip ASA ACE
Compounds (dosage 10 mg/kg)
Figure 1. Comparison of the anti-nociceptive effects of 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea and urea derivatives, non-steroidal anti-inflammatory
and analgesic drugs (dipyrone, ASA, and ACE) with control (C) group, at a concentration of 10 mg/kg, against acetic acid-induced pain in mice. Each group represents the mean
of six experiments and the vertical bars indicate the s.e.m. **p < 0.01, compared with the corresponding control value.

L. Santos et al. / Bioorg. Med. Chem. 16 (2008) 8526–8534
8529
mental model. These results seen in Figure 2 suggest that it is well
absorbed by the gastrointestinal tract.
the late phase is due to inflammation with a release of serotonin,
histamine, bradykinin, and prostaglandins45 and at least to some
degree, to the sensitization of central nociceptive neurons.44–46
In this model, compound 3 significantly inhibited both the first
and second phases by systemic route, in a dose-dependent way.
Analyzing some molecular properties and structural factors,
according to the method proposed by Lipinski,33,34 which evaluates
in silico oral bioavailability, it can be observed that compound 3
exhibited five of the proposed parameters, unlike the other com-
pounds of the series (4–12), which have infringed values of LogP
proposed by Lipinski’s’ rule (Table 3).33,34
Although compound 3 proved to be the most promising candi-
date for a future drug, it is important to point out that one violation
of the parameters proposed by Lipinski does not invalidate the bio-
logical potential of the molecules,34,35 since this method, in princi-
ple, indicates a probable oral bioavailability only by passive
transport. The excellent results for anti-nociceptive activity (intra-
peritoneally) suggest that compounds 4–9, 11, and 12 are not using
any other transport to the active site. However, further biological
assessments need to be conducted in order to identify possible
mechanisms that enable us to definitively state which type of
transport is being used by these compounds.
Acetic acid-induced pain in mice has the advantage that it al-
lows evidence to be obtained for effects produced by weak analge-
sics. Indeed, this test works not only for all major and minor
analgesics, but also for numerous other substances, including some
that have no analgesic action, for example, adrenergic blockers,36
anti-histamines,37 muscle relaxants,38 monoamine oxidase inhibi-
tors39 and neuroleptics.40 Nevertheless, because all analgesics inhi-
bit abdominal cramps, this method is useful for sifting molecules
whose pharmacodynamic properties are unknown.41,42 Compound
3 was therefore selected for more detailed studies in other models
of pain, in order to correlate, at least partially, the mechanism of
action of this compound.
The evaluation in the formalin test presents a distinctive bipha-
sic nociception response termed early and late phases. Drugs that
act primarily on the central nervous system inhibit both phases
equally, while peripherally acting drugs inhibit the late phase.43,44
The early phase is probably a direct result of the stimulation of
nociceptors in the paw, and reflects centrally mediated pain, while
The calculated ID50 values were 69.32 (55.9–85.9)
lmol/kg and
26.85 (23.0–31.3) mol/kg, with maximum inhibitions of 53.9
l
and 78.7% at 30 mg/kg, for the first and second phases, respectively
(Fig. 3). The ASA, an analgesic and anti-inflammatory drug which is
frequently used in therapeutic treatments, was inactive in prevent-
ing the first phase of the formalin-induced (neurogenic) pain, and
in the second phase it presented an ID50 value of 123.0 (77.0–
209.0) lmol/kg, compound 3 being about five times more potent.
In relation to dipyrone, which was active in both phases of this test,
compound 3 was much more active, being about two times more
active in the first phase, and 10 times more active in the late phase
than dipyrone itself, presenting ID50 values of 154.5 (99.0–238.8)
l
mol/kg and 263.7 (234.0–296.9) l
mol/kg, respectively.47 Another
interesting drug for comparison is the diclofenac, which presents a
better response in the formalin-induced pain test, and was less ac-
tive than compound 3. This drug presented, in this test, values of
>94
lmol/kg for the first phase and 34.5 (25.0–47.0) lmol/kg for
the second phase48
.
The capsaicin test in mice has been used to access the anti-noci-
ceptive effect of the tachykinin neurokinin-1 receptor antagonist,
the glutamate receptor antagonist, the nitric oxide (NO) synthase
inhibitor, and morphine.49 Compound 3, tested intraperitoneally
in mice at 10 mg/kg, was ineffective in this model (Fig. 4). This re-
sult suggests that it does not involve the participation of the vanil-
loid receptors, owing to the lack of analgesic effects in the
capsaicin model. The results support the hypothesis that the
anti-nociceptive mechanism differs largely regarding their action
on pain transmission in response to intraplantar injection of for-
malin or capsaicin although both formalin and capsaicin are
neurogenic.
It is important to mention that some known non-steroidal anti-
inflammatory drugs, including aspirin and acetaminophen, are
Table 3
Theoretical studies of solubility and permeability of 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea and urea derivatives by Lipinski’s rule of five
6"
NH
NH
5
6'
5"
4"
1"
2"
6
1
4
3
1'
2'
5'
4'
X
3'
3"
2
Cl
Y
O
Compound
X
Y
No. of atom
LogPa
MW
No. of ONb
No. of OHNHc
No. of rotb.d
PSAe
No. of viol.f
3
4
5
6
7
8
9
10
11
12
S
S
S
S
–H
4-Cl
3,4-Cl2
4-OCH3
4-CH3
–H
27
28
29
29
28
27
28
29
29
28
4.96
5.64
6.24
5.02
5.41
5.69
6.37
6.98
5.75
6.14
392.9
427.3
461.8
422.9
406.9
376.8
411.3
445.7
406.9
390.9
3
3
3
4
3
4
4
4
5
4
2
2
2
2
2
2
2
2
2
2
7
7
7
8
7
5
5
5
6
5
41.12
41.12
41.12
50.36
41.12
58.20
58.20
58.20
67.43
58.20
0
1
1
1
1
1
1
1
1
1
S
O
O
O
O
O
4-Cl
3,4-Cl2
4-OCH3
4-CH3
ASA
ACE
Dip.
—
—
—
—
—
—
13
11
20
1.43
0.68
ꢀ2.62
180.2
151.2
296.3
4
3
7
1
2
1
3
1
4
63.60
49.33
98.23
0
0
0
a
Method for LogP prediction developed at Molinspiration (miLogP2.2—November, 2005) is based on group contributions.
b
c
d
e
f
Sum of N and O H-bond acceptors.
Sum of NH and OH H-bond donors.
Number of rotatable bond.
Polar surface area.
Number of violations.

8530
L. Santos et al. / Bioorg. Med. Chem. 16 (2008) 8526–8534
First Phase
Second Phase
ID50 = 69.32 (55.92 - 85.94) µmol/kg
ID50 = 26.85 (23.06 - 31.29) µmol/kg
140
120
100
80
400
350
300
250
200
150
100
50
ns
37.8%
51%
**
53.9%
**
62.6%
**
60
**
78.7%
40
20
**
0
0
Control
6
10
30
Control
6
10
30
Compound 3 (mg/kg, i.p.)
Compound 3 (mg/kg, i.p.)
Figure 3. Effect of compound 1-(4-chlorophenyl)-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}thiourea (3), against formalin-induced pain in mice. Each column represents the
mean s.e.m. of six experimental values. nsNot significant. **p < 0.01.
100
90
80
70
60
50
40
30
20
10
0
ID50 = 62.09 (52.02 - 74.11) µmol/kg
ns
250
200
150
100
50
ns
42.6%
**
58.3%
**
0
Control
6
10
30
Compound 3 (mg/kg, i.p.)
Control
10
Figure 5. Effect of compound 1-(4-chlorophenyl)-3-{4-[(2E)-3-phenylprop-2-
enoyl]phenyl}thiourea (3), against glutamate-induced pain in mice. Each column
represents the mean s.e.m. of six experimental values. nsNot significant. **p < 0.01.
Compound 3 (mg/kg, i.p.)
Figure 4. Effect of compound 1-(4-chlorophenyl)-3-{4-[(2E)-3-phenylprop-2-
enoyl]phenyl}thiourea (3), against capsaicin-induced pain in mice, at 10 mg/kg,
ip. Each column represents the mean s.e.m. of six experimental values. nsNot
significant.
12
ns
10
8
ineffective or exhibit weak activity in the first phase of the forma-
lin test and in the capsaicin model, although they significantly in-
hibit the second phase of formalin-induced licking, compound 3
being the most powerful.
6
4
Compound 3 caused dose-related inhibition of the nociception
elicited by intraplantar glutamate (20
lmol/paw), with a calcu-
2
lated ID50 value of 62.1 (52.0–74.1) mol/kg and maximal inhibi-
l
tion of 58.3% at a dosage of 30 mg/kg (Fig. 5). These results
suggest that this compound acts inhibiting the release of neuro-
peptides from the sensory fibers, namely, NKs and kinins.50 The
current results corroborate this evidence, indicating a relevant
peripheral role in controlling the nociceptive processes.
0
C
10
Compound 3 (mg/kg, i.p.)
Figure 6. Effect of compound 1-(4-chlorophenyl)-3-{4-[(2E)-3-phenylprop-2-
enoyl]phenyl}thiourea (3), in the hot-plate test in mice, at 10 mg/kg, ip. Each
column represents the mean s.e.m. of six experimental values. nsNot significant.
When analyzed in the hot-plate method, compound 3 was not
capable of increasing the latency period of pain induced by heating
the plate at 10 mg/kg when administered intraperitoneally (Fig. 6).
This method has been designed to perform rapid and precise
screening of the central analgesic drugs.51 Morphine (26.6
lmol/
the hydrophobic (p) and electronic (a) effects of the substituents
kg, sc), used as a reference drug in this test, caused a significant
and marked analgesic effect.51 Our results suggest that the mecha-
nism by which compound 3 exerts analgesic activity does not in-
volve the participation of the opioid system, owing to the lack of
analgesic effects in the hot-plate test.
in the aromatic ring, and on a combination of both. The projected
order of potency of these five compounds for various parameter
dependencies is listed in Table 4. Comparison with the actual
experimentally determined potency order allows
a possible
deduction to be made concerning the probable operative param-
eters which in turn provides the basis for a new substituent
selection. Preliminary evaluation assessed with compounds
The manual procedure suggested by Topliss27 is based on the
assumption that the biological activity depends on the logic of

L. Santos et al. / Bioorg. Med. Chem. 16 (2008) 8526–8534
8531
Table 4
Potency order for various parameter dependencies27
Substituents
Parameters
a
p
2
p
ꢀ
p2
r
p
+
r
2
1
2–3
2–3
4
p
ꢀ
r
p
ꢀ
r
p
ꢀ 2
r
p
ꢀ 3
r
Es
3,4-Cl2
4-Cl
4-CH3
4-OCH3
H
1
2
3
4–5
4–5
1–2
1–2
3
4–5
4–5
1
2
4
5
3
1
2
3
5
4
1–2
3
1–2
4
3–4
3–4
1
2
5
5
3–4
1
2
3–4
2–5
2–5
2–5
2–5
1
5
5
a
Unfavorable steric effect from 4 substitution.
3–12 suggests that there is an unfavorable steric effect from sub-
stituent groups in the position 4; this suggestion could be seen
through the analysis of Table 4 as suggested by Topliss.27 Thus,
the two most potent members of these series, the 4-unsubstitut-
ed compounds 3 and 8, have the lowest steric requirements.
However, it is useful to suggest molecular modeling in order to
corroborate the influence of the steric effects of the substituent
groups in the position 4.
ing substituted aromatic aldehydes by the use of Claisen–Schmidt
condensation52 yielded five thioureas (3–7) and five ureas (8–12)
(Scheme 1).
4.2. General procedure for preparation of compounds
4.2.1. 1-(4-acetylphenyl)-3-(4-chlorophenyl)thiourea (1) and 1-
(4-acetylphenyl)-3-(4-chlorophenyl)urea (2)9
A
mixture of the corresponding 4-aminoacetophenone
(1.5 mmol) and 4-chlorophenylisothiocyanate or 4-chlorophenyl-
isocyanate (1.5 mmol) was dissolved in dry acetone (10 mL). The
mixture was stirred at room temperature for 3–6 h. The resulting
solids were collected on a filter and washed with dry acetone.
The resulting solid was crystallized from ethanol to yield 1-(4-
acetylphenyl)-3-(4-chlorophenyl)thiourea (1) and 1-(4-acetylphe-
nyl)-3-(4-chlorophenyl)urea (2) in pure form.
3. Conclusions
In summary, the synthetic method permitted the preparation of
10 chalcone derivatives with good to reasonable yields (45–90%).
Nine of these molecules (3–9, 11, and 12) presented anti-nocicep-
tive activity, with lower ID50 values than those obtained for the
positive control drugs (acetylsalicylic acid, acetaminophen, and
dipyrone). The structure–activity analysis shows that 1-phenyl-3-
{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea derivatives were
more potent than 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phe-
nyl}-urea derivatives, and there was an unfavorable steric effect
from four substituents. The compounds not substituted in ring B,
such as compounds 3 and 8, presented greater anti-nociceptive
activity. Compound 3 was a promising candidate for a future drug,
because it was the most potent in the series tested, being approx-
imately 47 times more active in the intraperitoneal treatment and
two times when treated orally, compared with the reference drugs.
In the formalin test, compound 3 significantly inhibited both the
first and second phases, by systemic route, in a dose-dependent
way, being more potent than diclofenac, a powerful anti-inflamma-
tory drug used in therapeutic treatments. It also presents activity
in the glutamate model. The mechanism by which this compound
exerts analgesic activity still remains undetermined, but our re-
sults suggest that it does not involve the participation of the opioid
system, or the vanilloid receptors, owing to the lack of analgesic ef-
fects in the hot-plate test and capsaicin model, respectively. The
procedure manual method suggested by Topliss suggests that
these compounds have an unfavorable steric effect from substitu-
tions at position 4. It is important to mention that no violation of
the Lipinski’s parameters was observed, characterizing compound
3 as drug-like compound.
4.2.2. 1-phenyl-3-{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-
thiourea and urea derivatives (3–12)9
Title compounds were prepared by reacting equivalent amounts
of 1-(4-acetylphenyl)-3-(4-chlorophenyl)thiourea (1) and 1-(4-
acetylphenyl)-3-(4-chlorophenyl)urea (2) with the corresponding
substituted aldehydes, in the presence of excess sodium hydroxide
(2.5 mmol) in dry methanol (5 mL). The mixture was stirred at
room temperature, and the resulting solids were collected in a
filter and washed three times with cold methanol. In most cases,
off-white to bright-yellow solids were formed within 2–20 h. The
product was recrystallized from the appropriate solvents (metha-
nol) where necessary.
4.3. Physicochemical data of the synthesized compounds
The melting points were determined with a Microquimica AP-
300 apparatus and are uncorrected. The IR spectra were recorded
with an Abb Bomen FTLA 2000 or Perkin-Elmer 720 spectrometer
on KBr disks. The NMR (1H and 13C NMR) spectra were recorded
on
a Brucker Ac-200 F (300 MHz) or Varian Oxford AS-400
(400 MHz) instrument, using tetramethylsilane as an internal stan-
dard. Elementary analysis was carried out using a Perkin-Elmer
2400. The percentages of elements determined (C, H, and N) were
in agreement with the product formula (within 0.4% of theoretical
values for C). The solvents and reagents were purified in the usual
manner where necessary.
4. Experimental
4.1. Synthesis
4.3.1. 1-(4-Acetylphenyl)-3-(4-chlorophenyl)thiourea (1)
A
procedure based on Claisen–Schmidt condensation was
Yield 45%; mp 168–169 °C (from ethanol); IR (KBr) mmax 3478
developed for the syntheses of all the 1-phenyl-3-{4-[(2E)-3-phe-
nylprop-2-enoyl]phenyl}-thiourea and urea derivatives. The syn-
theses of acetophenone derivatives (1–2) were obtained by
reaction of p-amino acetophenone with the corresponding 4-chlor-
ophenylisothiocyanate or 4-chlorophenyl-isocyanate derivatives.
The subsequent treatment of these derivatives with solid sodium
hydroxide, in methanol, at room temperature, and the correspond-
(NH), 1656 (CO) cmꢀ1 1H NMR (300 MHz, DMSO-d6) d 2.53 (s,
;
3H, COMe), 7.38 (d, 2H, H300-500, J = 8.80 Hz), 7.52 (d, 2H, H200-600,
J = 8.80 Hz), 7.68 (d, 2H, H20-60, J = 8.80 Hz), 7.92 (d, 2H, H30-50,
J = 8.80 Hz), 10.11 (br s, 1H, NH), 10.19 (br s, 1H, NH); 13C NMR
(300 MHz, DMSO-d6) d 26.47 (COMe), 121.77 (C20-60), 125.23
(C200-600), 128.35 (C30-50), 128.52 (C400), 128.90 (C300-500), 132.22
(C40), 138.14 (C100), 143.92 (C10), 179.40 (CS(NH)2), 196.58 (CO).

8532
L. Santos et al. / Bioorg. Med. Chem. 16 (2008) 8526–8534
4.3.2. 1-(4-Acetylphenyl)-3-(4-chlorophenyl)urea (2)
55.35 (OMe), 114.39 (C3-5), 119.42 (Ca
), 121.85 (C20-60), 125.25
Yield 45%; mp 223–225 °C (from ethanol); IR (KBr) mmax 3478
(C200-600), 127.39 (C400), 128.37 (C2-6), 128.45 (C40), 129.18 (C300-
500), 130.68 (C30-50), 133.14 (C1), 138.22 (C100), 143.45 (Cb),
143.83 (C10), 161.28 (C4), 179.36 (CS(NH)2), 187.51 (CO).
(NH), 1712 (CS(NH)2), 1654 (CO) cmꢀ1 1H NMR (300 MHz,
,
DMSO-d6) d 2.53 (s, 3H, COMe), 7.33 (d, 2H, H300-500, J = 8.80 Hz),
7.50 (d, 2H, H200-600, J = 8.80 Hz), 7.58 (d, 2H, H20-60, J = 8.80 Hz),
7.90 (d, 2H, H30-50, J = 8.80 Hz), 8.93 (br s, 1H, NH), 9.12 (br s, 1H,
C23H19ClN2O2S requires: C (65.32%) H (4.53%) N (6.62%). Found: C
(65,00%) H (4,55%) N (6,68%).
13
NH); C NMR (300 MHz, DMSO-d6) d 26.32 (COMe), 117.25 (C20-
60), 119.97 (C200-600), 125.77 (C400), 128.66 (C30-50), 129.62 (C300-
500), 130.57 (C40), 138.31 (C100), 144.18 (C10), 152.09 (CO(NH)2),
196.29 (CO).
4.3.7. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(4-methylphenyl)prop-
2-enoyl]-phenyl}thiourea (7)
Yield 37%; mp 208–210 °C (from methanol); IR (KBr)
(NH) 1640 (CO) cmꢀ1 1H NMR (300 MHz, DMSO-d6) d 2.35 (s, 3H,
Me), 7.21 (d, 2H, H3-5, J = 8.36 Hz), 7.25 (d, 2H, H300-500, J = 8.36 Hz),
mmax 3454
;
4.3.3. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-phenylprop-2-
enoyl]phenyl}thiourea (3)
7.27 (d, 2H, H2-6, J = 8.36 Hz), 7.65 (d, 1H, Ha, J = 15.38 Hz), 7.73
Yield 50%; mp 178–180 °C (from methanol); IR (KBr)
m
max 3453
(d, 2H, H200-600, J = 8.36 Hz), 7.76 (d, 2H, H20-60, J = 8.36 Hz), 7.89
(d, 1H, Hb, J = 15.38 Hz), 8.01 (d, 2H, H30-50, J = 8.36 Hz), 9.45 (br
s, 2H, NH); 13C NMR (300 MHz, DMSO-d6) d 21.06 (Me), 120.89
(NH), 1640 (CO) cmꢀ1; 1H NMR (300 MHz, DMSO-d6) d 7.40 (d, 2H,
H3-5, J = 8.55 Hz), 7.53 (d, 2H, H300-500, J = 8.55 Hz), 7.68 (d, 1H, H
a,
J = 15.87 Hz), 7.74 (m, 4H, H2-6, H200-600), 7.87 (dd, 1H, H4,
J = 8.55 Hz), 8.06 (d, 1H, Hb, J = 15.87 Hz), 8.18 (d, 2H, H20-60,
J = 8.86 Hz), 8.29 (d, 2H, H30-50, J = 8.86 Hz), 10.15 (br s, 1H, NH),
10.24 (br s, 1H, NH); 13C NMR (300 MHz, DMSO-d6) d 121.76
(Ca
), 121.70 (C20-60), 123.61 (C200-600), 124.71 (C400), 128.49 (C2-
6), 128.81 (C300-500), 129.21 (C3-5), 129.50 (C30-50), 132.04 (C40),
132.79 (C1), 139.18 (C4), 140.51 (C100), 143.45 (C10), 144.15 (Cb),
179.27 (CS(NH)2), 187.56 (CO). C23H19ClN2OS requires: C (67.89%)
H (4.71%) N (6.88%). Found: C (66.98%) H (4.50%) N (6.92%).
(C20-60), 124.04 (C ), 125.25 (C200-600), 128.40 (C2-6), 129.10 (C4),
a
129.50 (C3-5), 130.05 (C400), 130.94 (C300-500), 131.81 (C30-50),
132.56 (C40), 135.68 (C1), 138.17 (C100), 140.39 (C10), 144.29 (Cb),
179.36 (CS(NH)2), 187.36 (CO). C22H17ClN2OS requires: C (67.25%)
H (4.36%) N (7.13%). Found: C (67.15%) H (4.16%) N (7.00%).
4.3.8. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-phenylprop-2-enoyl]-
phenyl}urea (8)
Yield 76%; mp 202–204 °C (from methanol); IR (KBr)
m
;
max 3323
(NH) 1712 (CO urea) 1652 (CO
a
,b-unsaturated) cmꢀ1
1H NMR
4.3.4. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(4-chlorophenyl)prop-2-
enoyl]-phenyl}thiourea (4)
(300 MHz, DMSO-d6) d 7.31 (d, 2H, H3,5, J = 8.95 Hz), 7.45 (m,
3H, H300-500, H4), 7.53 (d, 2H, H2-6, J = 8.95), 7.67 (d, 2H, H200-600,
Yield 90%; mp 187–190 °C (from methanol); IR (KBr)
m
max 3453
J = 8.95), 7.71 (d, 1H, Ha
, J = 15.79 Hz), 7.87 (dd, 2H, H20-60,
(NH) 1654 (CO) cmꢀ1; 1H NMR (300 MHz, DMSO-d6) d 7.16 (d, 2H,
H300-500, J = 8.65 Hz), 7.24 (d, 2H, H3-5, J = 8.65 Hz), 7.50 (d, 4H, H200-
J = 8.95 Hz), 7.93 (d, 1H, Hb, J = 15.79 Hz), 8.12 (d, 2H, H30-50,
J = 8.95 Hz), 9.52 (br s, 2H, NH); 13C NMR (300 MHz, DMSO-d6) d
600 and H2-6, J = 8.65 Hz), 7.64 (d, 1H, H
a
, J = 15.38 Hz), 7.91 (d, 2H,
117.40 (C20-60), 119.97 (C200-600), 122.02 (C
a), 125.60 (C4), 128.58
H20-60, J = 8.65 Hz), 7.95 (d, 1H, Hb, J = 15.38 Hz), 8.00 (d, 2H, H30-
50, J = 8.65 Hz), 9.96 (br s, 2H, NH); 13C NMR (300 MHz, DMSO-d6)
(C2-6), 128.72 (C3-5), 128.87 (C300-500), 130.02 (C30-50), 130.37
(C400), 130.94 (C40), 134.82 (C1), 138.51 (C100), 142.99 (C10),
144.61 (Cb), 152.29 (CO(NH)2), 187.28 (CO). C22H17ClN2O2 re-
quires: C (70.12%) H (4.55%) N (7.43%). Found: C (69,80%) H
(4,49%) N (7.22%).
d 122.83 (Ca
), 123.23 (C20-60), 127.45 (C200-600), 127.74 (C2-6),
128.43 (C400), 128.87 (C3-5), 129.33 (C300-500), 129.99 (C40), 130.28
(C30-50), 131.15 (C4), 134.06 (C1), 134.53 (C100), 140.51 (C10 and
Cb), 179.50 (CS(NH)2), 187.73 (CO). C22H16Cl2N2OS requires: C
(61.83%) H (3.77%) N (6.56%). Found: C (61.90%) H (3.80%) N
(6.50%).
4.3.9. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(4-chlorophenyl)prop-2-
enoyl]-phenyl}urea (9)
Yield 87%; mp 249–251 °C (from methanol); IR (KBr) max 3334
m
4.3.5. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(3,4-
(NH) 1710 (CO urea) 1640 (CO ;
a
,b-unsaturated) cmꢀ1 1H NMR
dichlorophenyl)prop-2-enoyl]-phenyl}thiourea (5)
(300 MHz, DMSO-d6) d 7.34 (d, 2 H, H3-5, J = 8.80 Hz), 7.51 (d,
Yield 80%; mp 172–174 °C (from methanol); IR (KBr)
(NH) 1642 (CO) cmꢀ1 1H NMR (300 MHz, DMSO-d6) d 7.40 (d, 2H,
H300-500, J = 8.25 Hz), 7.55 (d, 1H, H6, J = 8.25 Hz), 7.68 (d, 1H, H
mmax 3451
2H, H300-500, J = 8.80 Hz), 7.53 (d, 2H, H2-6), 7.63 (d, 2H, H200-600),
;
7.70 (d, 1H, Ha
, J = 15.85 Hz), 7.92 (d, 2H, H20-60, J = 8.80 Hz),
a,
8.02 (d, 1H, Hb, J = 15,85 Hz), 8.14 (d, 2H, H30-50, J = 8.80 Hz), 9.03
J = 15.6 Hz), 7.72 (d, 2H, H200-600, J = 8.25 Hz), 7.76 (d, 2H, H20-60,
J = 8.25 Hz), 7.85 (d, 1H, H5, J = 8.25 Hz), 8.04 (d, 1H, Hb,
J = 15.6 Hz), 8.17 (d, 2H, H30-50, J = 8.25 Hz), 8.26 (s, 1H, H2),
10.16 (br s, 1H, NH), 10.25 (br s, 1H, NH); 13C NMR (300 MHz,
(br s, 1H, NH), 9.25 (br s, 1H, NH); 13C NMR (300 MHz, DMSO-d6)
d
117.37 (C20-60), 120 (C ), 122.77 (C200-600), 125.77 (C2-6),
a
128.63 (C4), 128.89 (C3-5), 130.14 (C400), 130.45 (C300-500), 130.97
(C30-50), 130.80 (C40), 134.84 (C1), 138.28 (C100), 141.58 (C10),
144.41 (Cb), 152.09 (CO(NH)2), 187.19 (CO). C22H16Cl2N2O2 re-
quires: C (64.25%) H (3.92%) N (6.81%). Found: C (64.15%) H
(3.50%) N (6.78%).
DMSO-d6) d 121.82 (C20-60), 124.07 (C ), 125.28 (C200-600), 128.35
a
(C6), 128.43 (C300-500), 129.13 (C2), 129.53 (C30-50), 130.08 (C400),
130.97 (C5) 131.32 (C40), 131.84 (C4), 132.59 (C3), 135.68 (C1),
138.20 (C100), 140.62 (Cb), 144.32 (C10), 179.39 (CS(NH)2), 187.39
(CO). C22H15Cl3N2OS requires: C (57.22%) H (3.27%) N (6.07%).
Found: C (57.30%) H (3.50%) N (6.10%).
4.3.10. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(3,4-
dichlorophenyl)prop-2-enoyl]-phenyl}urea (10)
Yield 87%; mp 198-200 °C (from methanol); IR (KBr) mmax 3343
4.3.6. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(4-
methoxyphenyl)prop-2-enoyl]-phenyl}thiourea (6)
(NH) 1713 (CO urea) 1641 (CO
(300 MHz, DMSO-d6) d 7.35 (d, 2H, H300-500, J = 8.83 Hz), 7.51 (d,
2H, H200-600, J = 8.83 Hz), 7.63 (d, 2H, H20-60), 7.67 (d, 1H, H
a,
a ;
,b-unsaturated) cmꢀ1 1H NMR
Yield 70%; mp 195–197 °C (from methanol); IR (KBr)
(NH) 1638 (CO) cmꢀ1 1H NMR (300 MHz, DMSO-d6) d 3.82 (s, 3H,
OMe), 7.02 (d, 2H, H3-5, J = 8.86 Hz), 7.40 (d, 2H, H300-500,
m
max 3453
;
J = 15.44 Hz), 7.70 (d, 1H, H6, J = 8.83 Hz), 7.86 (d, 1H, H5,
J = 8.83 Hz), 8.04 (d, 1H, Hb, J = 15.44 Hz), 8.16 (d, 2H, H30-50,
J = 8.83 Hz), 8.26 (s, 1H, H2), 9.12 (br s, 2H, NH); 13C NMR
(300 MHz, DMSO-d6) d 117.34 (C20-60), 120.00 (C200-600), 124.10
J = 8.86 Hz), 7.54 (d, 2H, H2-6), 7.70 (d, 1H, Ha, J = 15.75 Hz), 7.73
(d, 2H, H200-600, J = 8.86 Hz), 7.82 (d, 1H, Hb, J = 15.75 Hz), 7.86 (d,
2H, H20-60, J = 8.86 Hz), 8.13 (d, 2H, H30-50, J = 8.86 Hz), 10.18 (br
s, 1H, NH), 10.27 (br s, 1H, NH); 13C NMR (300 MHz, DMSO-d6) d
(C
a
), 125.75 (C6), 128.63 (C300-500), 129.01 (C2), 130.02 (C400),
130.22 (C30-50), 130.83 (C5), 130.92 (C40), 131.78 (C4), 132.48

L. Santos et al. / Bioorg. Med. Chem. 16 (2008) 8526–8534
8533
(C3), 135.74 (C1), 138.28 (C100), 140.22 (Cb), 144.55 (C10), 152.09
(CO(NH)2), 187.02 (CO). C22H15Cl3N2O2 requires: C (59.28%) H
(3.39%) N (6.28%). Found: C (59.30%) H (3.40%) N (6.18%).
control animals were pretreated with compounds 1-phenyl-3-{4-
[(2E)-3-phenylprop-2-enoyl]phenyl}-thiourea and urea derivatives
or standard drugs. For compound 3, the abdominal constriction re-
sponse was analyzed, following pretreatment via oral route, of a
dosage of 50–200 mg/kg.
4.3.11. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(4-
methoxyphenyl)prop-2-enoyl]-phenyl}urea (11)
Yield 88%; mp 208–210 °C (from methanol); IR (KBr)
m
;
max 3343
1H NMR
4.4.3. Formalin test
(NH) 1708 (CO urea) 1641 (CO
a
,b-unsaturated) cmꢀ1
The observation chamber was a glass cylinder of 20 cm in diam-
eter, with a mirror at a 45° angle to allow clear observation of the
animal’s paws. The mice were treated with 0.9% saline solution (ip)
or compound 3 (6–30 mg/kg, ip), 30 min before formalin injection.
Each animal was placed in the chamber for 5 min before treatment,
in order to acclimatize to the new environment. The formalin test
was carried out as described by Hunskaar and Hole,56 with minor
modifications.55,57 Twenty microliters of a 2.5% formalin solution
(0.92% formaldehyde) in 0.9% saline solution were injected intra-
plantarly 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 (0–5 and 15–30 min after formalin injec-
tion, respectively).
(300 MHz, DMSO-d6) d 3.81 (s, 3H, OMe), 7.00 (d, 2H, H3-5,
J = 8.95 Hz), 7.32 (d, 2H, H300-500, J = 8.95 Hz), 7.52 (d, 2H, H2-6,
J = 8.95), 7.64 (d, 2H, H200-600, J = 8.95 Hz), 7.68 (d, 1H,
Ha,
J = 15.26 Hz), 7.78 (d, 1H, Hb, J = 15.26 Hz), 7.83 (d, 2H, H20-60,
J = 8.42 Hz), 8.11 (d, 2H, H30-50, J = 8.42 Hz), 9.29 (br s, 2H, NH);
13C NMR (300 MHz, DMSO-d6) d 55.32 (OMe), 114.36 (C3-5),
117.34 (C20-60), 119.48 (C ), 119.97 (C200-600), 125.66 (C400),
a
127.45 (C40), 128.61 (C2-6), 129.88 (C300-500), 130.60 (C30-50),
131.29 (C1), 138.43 (C100), 143.02 (Cb), 144.23 (C10), 152.18 (C4),
161.19 (CO(NH)2), 187.19 (CO). C23H19ClN2O3 requires:
C
(67.90%) H (4.71%) N (6.89%). Found: C (67.20%) H (4.22%) N
(6.92%).
4.3.12. 1-(4-Chlorophenyl)-3-{4-[(2E)-3-(4-methylphenyl)prop-
2-enoyl]-phenyl}urea (12)
Yield 93%; mp 217–219 °C (from methanol); IR (KBr)
m
;
max 3343
4.4.4. Capsaicin-induced nociception
(NH) 1709 (CO urea) 1643 (CO
a
,b-unsaturated) cmꢀ1
1H NMR
The procedure used was similar to that described previously.49
(300 MHz, DMSO-d6) d 2.34 (s, 3H, Me), 7.26 (d, 2H, H3-5,
After the adaptation period, 20 ml of capsaicin (1.6 lg/paw) was
J = 8.83 Hz), 7.31 (d, 2H, H2-6, J = 8.83 Hz), 7.51 (d, 2H, H300-500),
injected intraplantarly into the right hindpaw. 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 nociception.
The animals were treated with compound 3 via ip (10 mg/kg)
30 min prior to capsaicin injection. The control animals received
a similar volume of saline solution, intraperitoneally.
7.66 (d, 2H, H200-600, J = 8.83 Hz), 7.68 (d, 1H, H
a, J = 16.18 Hz),
7.75 (d, 2H, H20-60, J = 8.83 Hz), 7.88 (d, 1H, Hb, J = 16.18 Hz),
8.10 (d, 2H, H30-50, J = 8.83 Hz), 9.65 (br s, 2H, NH); 13C NMR
(300 MHz, DMSO-d6) d 21.03 (Me), 117.37 (C20-60), 119.97 (C200-
600), 120.95 (C ), 125.54 (C400), 128.55 (C2-6), 128.75 (C300-500),
a
129.50 (C3-5), 129.93 (C30-50), 131.03 (C40), 132.10 (C1), 138.60
(C4), 140.39 (C100), 143.05 (C10), 144.55 (Cb), 152.32 (CO(NH)2),
187.25 (CO). C23H19ClN2O2 requires: C (70.68%) H (4.90%) N
(7.17%). Found: C (70,20%) H (4,70%) N (7.00%).
4.4.5. Hot-plate test
The hot-plate test was used to measure response latencies,
according to the method described by Eddy and Leimback.58 The
mice were treated with saline solution, morphine (10 mg/kg, sc)
or compound 3 (10 mg/kg, ip), and placed individually on a hot-
plate maintained at 56 1 °C. The time between placing the animal
on the hot-plate and the occurrence of either the 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 be-
fore the assay.
4.4. Biological assay
4.4.1. Animals
Swiss mice (25–35 g) were obtained from the Animal House of
the University of Vale do Itajaí (Itajaí, Brazil). They were housed in
automatically controlled temperature conditions (23 2 °C and
12 h light–dark cycles). The animals were given access to water
and Nuvital chow ad libitum, unless otherwise indicated. They re-
mained in the appropriate laboratory at UNIVALI until several
hours before the experiments. The allocation of animals into the
different groups was randomized, and the experiments were car-
ried out in blind conditions. Since some suffering might result from
experiments, the IASP Committee for Research and Ethical Issues
Guidelines53 were followed.
4.4.6. Glutamate-induced nociception
The animals were treated with compound 3 via ip (6–30 mg/kg)
30 min before the glutamate injection. A volume of 20
ll of gluta-
mate solution (30 mol/paw), made up in phosphate-buffered sal-
l
ine (PBS), was injected intraplantarly under the surface of the right
hind paw, as described previously by Beirith.48 After injection with
glutamate, the animals were individually placed into glass cylin-
ders of 20 cm in diameter and observed from 0 to 15 min. The time
spent licking or biting the injected paw was timed with a chronom-
eter and considered as indicative of pain.
4.4.2. Acetic acid-induced writhing
The abdominal constriction response caused by intraperitoneal
injection of diluted acetic acid (0.6%), was carried out according to
the procedures described previously54,55 with minor modifications.
The animals were pretreated with 1-phenyl-3-{4-[(2E)-3-phenyl-
prop-2-enoyl]phenyl}-thiourea derivatives (3–7) or 1-phenyl-3-
{4-[(2E)-3-phenylprop-2-enoyl]phenyl}-urea derivatives (8–12)
(1–60 mg/kg, ip) or standard drugs 30 min before acetic acid infec-
tion. The control animals received a similar volume of 0.9% NaCl
(10 ml/kg, ip). All the experiments were carried out at 20–22 °C.
After the challenge, each animal was placed in a separate glass fun-
nel, and the number of abdominal contractions of the abdominal
muscles together with stretching of the hind limbs was cumula-
tively counted over a period of 20 min. Anti-nociceptive activity
4.4.7. Statistical analysis
The results are presented as means SEM, except for the mean
ID50 values (i.e. the dose of drugs or compounds reducing the anti-
nociceptive responses by 50% relative to the control value) which
are reported as geometric means accompanied by their respective
95% confidence limits. The statistical significance between the
groups was determined by analysis of variance followed by Dun-
nett’s multiple comparison test. P-values of less than 0.05 were

8534
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