Synthesis and pharmacological evaluation of N-phenyl-acetamide sulfonamides designed as novel non-hepatotoxic analgesic candidates

Article abstract of DOI:10.1016/j.ejmech.2009.02.026

In this paper we report the design, synthesis and pharmacological evaluation of a series of N-phenyl-acetamide sulfonamide derivatives (5a-g), planned by structural modification on the prototype paracetamol (1). In this series (5a-g), compound LASSBio-1300 (5e; ID₅₀ = 5.81 μmol/kg) stands out as a new non-hepatotoxic analgesic drug candidate. The increase of area, volume and eletrostatic potential of paracetamol's analogues seems to be beneficial to the analgesic activity. Unlike paracetamol (1) and the other analogues (5a, 5d-g), compounds 5b and 5c presented an important anti-hypernociceptive activity associated to inflammatory pain.

Full text of DOI:10.1016/j.ejmech.2009.02.026

European Journal of Medicinal Chemistry 44 (2009) 3612–3620
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of N-phenyl-acetamide sulfonamides
designed as novel non-hepatotoxic analgesic candidates
a
,
Maria Letıcia de Castro Barbosa b, Gabriela Muniz de Albuquerque Melo c, Yolanda Karla Cupertino da
´
Silva c, Raquel de Oliveira Lopes a , Everton Tenorio de Souza , Aline Cavalcanti de Queiroz ,
,
b
c
c
´
Salete Smaniotto d, Magna Suzana Alexandre-Moreira c , Eliezer J. Barreiro , Lıdia Moreira Lima
,
a,
b
a,b,
**
*
´
a LASSBio1 – Laboratorio de Avaliaçao e Sıntese de Substancias Bioativas, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, P.O. Box 68024, 21944-971
´
˜
´
ˆ
´
Rio de Janeiro, RJ, Brazil
b
´
˜
´
´
Pos-graduaçao em Quımica, Instituto de Quımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c LaFI – Laborato´rio de Farmacologia e Imunidade, Instituto de Cieˆncias Biolo´gicas e da Sau´de, Universidade Federal de Alagoas, Maceio´, AL, Brazil
d Laborato´rio de Imunohistologia – Setor de Histologia, Instituto de Cieˆncias Biolo´gicas e da Sau´de, Universidade Federal de Alagoas, Maceio´, AL, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper we report the design, synthesis and pharmacological evaluation of a series of N-phenyl-
acetamide sulfonamide derivatives (5a–g), planned by structural modification on the prototype
Received 2 December 2008
Received in revised form
23 February 2009
Accepted 25 February 2009
Available online 4 March 2009
paracetamol (1). In this series (5a–g), compound LASSBio-1300 (5e; ID50 ¼ 5.81
mmol/kg) stands out as
a new non-hepatotoxic analgesic drug candidate. The increase of area, volume and eletrostatic potential
of paracetamol’s analogues seems to be beneficial to the analgesic activity. Unlike paracetamol (1) and
the other analogues (5a, 5d–g), compounds 5b and 5c presented an important anti-hypernociceptive
activity associated to inflammatory pain.
Keywords:
Analgesic
Ó 2009 Elsevier Masson SAS. All rights reserved.
LASSBio-1300
N-Phenyl-acetamide sulfonamides
Paracetamol
Hepatotoxicity
1. Introduction
The mechanism of paracetamol’s hepatotoxicity is well known
and is directly associated to its hepatic metabolism. It is primarily
Paracetamol (acetaminophen, 4-hydroxyacetanilide, 1) was
synthesized in 1878 and the first clinical use was reported in 1893,
but only after it was identified as the active metabolite of phen-
acetin (2) and acetanilide (3) that it was marketed as a drug [1].
Introduced worldwide in the 1950s as an antipyretic and
analgesic drug, paracetamol (1) is still one of the most popular
over-the-counter drugs, which is frequently used on a prescription
basis for the relief of acute and chronic pain. Within the thera-
peutic range, it is, usually, well tolerated, with few side effects.
However, when taken in over dosage, paracetamol (1) may cause
severe and sometimes fatal hepatic necrosis [2,3].
metabolized by the liver, being 90% eliminated as glucoronide and
sulphate metabolites, 5% excreted unchanged and 5% oxidized, by
cytochrome P4502E1 (CYP2E1), to N-acetyl-p-benzoquinone imine
(NAPQI) (Scheme 1) [4].
Hepatotoxicity arises only through NAPQI (4), a highly reactive
compound that normally conjugates with glutathione, being
eliminated as an inactive metabolite. However, in conditions
where the production of NAPQI exceeds that of glutathione, this
reactive metabolite binds covalently to liver proteins and causes
dose-related liver injury by centrilobular necrosis [5,6].
In a continuing effort to develop new analgesic drug candidates,
we report in this paper the design, synthesis and pharmacological
evaluation of N-phenyl-acetamide sulfonamide derivatives (5a–g),
planned by structural modification on the prototype paracetamol (1).
The design concept considered the need to carry out structural
modifications in the toxicophoric unit of paracetamol (1), avoiding
its biotransformation to the reactive metabolite NAPQI (4).
The structural design of this new series was accomplished first
by applying a non-classical bioisosterism [7], represented by the
* Corresponding author.
** Corresponding author. Po´s-graduaça˜o em Quı´mica, Instituto de Quı´mica, Uni-
versidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Tel.: þ55 2125626503;
fax: þ55 2125626644.
E-mail addresses: msamoreira@yahoo.com (M.S. Alexandre-Moreira), lidia@
pharma.ufrj.br (L.M. Lima).
1
http://www.farmacia.ufrj.br/lassbio/.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.026

M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
3613
O
O
O
HN
CH3
HN
CH3
HN
CH3
CYP450
CYP450
CH3
OH
O
phenacetin (2)
paracetamol (1)
acetanilide (3)
CYP2E1
90
O
5
5
O
O
N
CH3
HN
CH3
O
HN
CH3
HN
CH3
O
OGlicuronide
NAPQI (4)
OSulphate
OH
Scheme 1.
replacement of the phenolic hydroxyl group (subunit B) by a non-
oxidizable one, comprised by the sulfonamide subunit (Chart 1).
Then, the introduction of an ethyl side chain was performed, since it
is also present in the structure of the analgesic phenacetin (2),
linked to the nitrogen of the sulfonamide moiety, in a classical
example of the homologation strategy (Chart 1) [7]. Moreover, in
order to investigate the importance of stereoelectronic and
conformational parameters to the analgesic activity, the nature of
the sulfonamide moiety (subunit C) was modified by the intro-
duction of diethyl (5c), piperidine (5d), piperazine (5d), N-methyl-
piperazine (5e), morpholine (5f) and thiomorpholine (5g) units
(Chart 1).
2. Results and discussion
2.1. Chemistry
The synthesis of these new series was undertaken using a clas-
sical methodology, based on several functional group interconver-
sions, employing the sulfonyl chloride derivative (7) as the key
intermediate. This compound could be easily obtained, in high
yield, by the synthetic sequence depicted in Scheme 2. The acety-
lation of aniline (6) with acetic anhydride, furnished the acetanilide
(3) in 90% yield [8]. The second step in the synthesis of 7 was based
on a regioselective electrophilic aromatic substitution employing
chlorosulfonic acid, obtaining 7 in 85% yield [9,10]. With this key
intermediate in hands, the target compounds could be prepared by
condensation of the 4-(acetylamine)benzenesulfonyl chloride (7)
with functionalized amines to give the desired sulfonamide deriv-
atives (5a–g) in 50–70% yield, as shown in Scheme 2 [11].
2.2. Analgesic and hypothermic activities
The analgesic activity of N-phenyl-acetamide sulfonamide
derivatives (5a–g) was initially performed employing the acetic
Table 1
Effect of derivatives 5a–g (100
mmol/kg, p.o.), and paracetamol (100 mmol/kg, p.o.)
on acetic acid-induced writhing in mice.
Substance
n
Writhing number mean ꢁ S.E.M.a
% of inhibition
Control
8
8
8
8
8
8
8
8
8
43.2 ꢁ 1.7
–
1 (Paracetamol)
5a (LASSBio-1301)
5b (LASSBio-1298)
5c (LASSBio-1297)
5d (LASSBio-1299)
5e (LASSBio-1300)
5f (LASSBio-1296)
5g (LASSBio-1295)
28.5 ꢁ 3.2*
22.2 ꢁ 4.1**
23.4 ꢁ 4.5**
30.4 ꢁ 2.5*
16.8 ꢁ 3.1**
10.8 ꢁ 1.7**
24.5 ꢁ 3.7**
24.2 ꢁ 3.5**
34.0%
48.6%
45.8%
29.6%
61.1%
75.0%
43.2%
44.0%
Scheme 2. Reagents and conditions: a) acetic anhydride, glacial acetic acid, CH3CO2Na
anhydrous, r.t., 30 min, 90%; b) HSO3Cl, 60 ꢀC, 30 min, 85%; c) functionalized amines,
CH2Cl2, r.t., 30 min, 50–70%.
a
The asterisks denote the significance levels in comparison with control groups,
*P < 0.05 and **P < 0.01.

3614
M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
other NSAIDs (non-steroidal anti-inflammatory drugs) have no
effect [17–19].
40
30
Paracetamol
As shown in Table 2, the oral administration of compounds 5g
(LASSBio-1295) and 5e (LASSBio-1300) and paracetamol increased
significantly the reaction time to the nociceptive response in the
hot-plate test, while morphine induced a marked increase in the
latency of the animals at (9.0 ꢁ 1.4 s) 60 min and (7.5 ꢁ 0.5 s)
90 min. No significant activity was found for compounds 5a–f
(Table 2). The observations that 5g (LASSBio-1295), 5e (LASSBio-
1300) and paracetamol (1) increased the baseline of animals in the
hot-plate model suggest that these compounds present supra-
spinal analgesic activity.
The anti-hyperalgesic activity of N-phenyl-acetamide sulfon-
amide derivatives (5a–g) and paracetamol (1) was determined
using the formalin test [20,21]. Formalin is known to produce
biphasic pain behaviors. The first transient phase is ascribed to the
direct effect of formalin on sensory C fibers, and the second pro-
longed phase is associated to the development of an inflammatory
response and the release of algesic mediators [20–23]. The results
obtained, depicted in Fig. 2, show a significant antinociceptive
response observed in the first phase (i.e. central phase) of the
5e (LASSBio-1300)
20
10
0
0.0
0.5
1.0
1.5
2.0
2.5
Log dose (µmol/kg)
Fig. 1. Dose–response curves of the antinociceptive effect of paracetamol (-) (100, 30,
10, and 3 mol/kg) and 5e (LASSBio-1300) (B) (100, 30, 10, and 3 mol/kg) on the 0.6%
m
m
acetic acid-induced abdominal constrictions observed in a period of 25 min in mice.
Data are expressed as the total number writhings calculated for 8 animals. *P < 0.05,
ANOVA followed by Dunnett’s test.
formalin-induced pain after oral administration (100
mmol/kg) of
acid-induced abdominal writhing model in mice [12–14], using
paracetamol (1) as standard compound.
In this model the N-phenyl-acetamide sulfonamide derivatives
(5a–g) and paracetamol (1) were evaluated at the dose of 100 mmol/
paracetamol (1), 5f (LASSBio-1296) and 5d (LASSBio-1299). On the
other hand, the analysis of the second phase of the formalin test (i.e.
inflammatory phase) indicated an anti-hyperalgesic activity only
for compounds 5c (LASSBio-1297) and 5b (LASSBio-1298).
kg, by oral administration. The results, illustrated in Table 1,
demonstrated that all derivatives (5a–g) produced marked inhibi-
tion of acetic acid-induced writhing response, compounds 5d
(LASSBio-1299; 61.1%) and 5e (LASSBio-1300; 75.0%) being signifi-
cantly more active than the standard paracetamol (34.0%) in the
Considering the hypothermic action, in mice, of paracetamol (1)
and its derivatives [24], which seems to be mediated by inhibition
of prostaglandin endoperoxide synthase 1 gene-derived protein
(i.e. COX-3) [25,26], we investigated the ability of this prototype (1)
and its sulfonamide analogues 5a–g to interfere on the body
temperature, using the test behavioral measures of rectal temper-
ature in mice [27]. In this model, the temperature was measured
with room temperature of 25 ꢀC. After 1 h of acclimatization, the
baseline temperature was determined. The same was measured
before and after administration of test compounds (5a–g) and
screening dose of 100
selected in order to study its dose–response curve. As depicted in
Fig. 1, paracetamol (dose ¼ 100.0, 30.0, 10.0, and 3.0 mol/kg) and
LASSBio-1300 (dose ¼ 100.0, 30.0, 10.0, and 3.0 mol/kg) produced
dose-related inhibition of acetic acid-induced abdominal
constrictions in mice, with ID50 values of 19.27 mol/kg and
5.81 mol/kg, respectively.
In order to determine an eventual central antinociceptive
activity, compounds 5a–g (100 mol/kg, p.o.) were also evaluated
in the hot-plate test [15], using morphine (15 mol/kg, i.p.) and
paracetamol (100 mol/kg, p.o.) as standards. In this model, the
mmol/kg, p.o. Thus, LASSBio-1300 (5e) was
m
m
a
m
paracetamol (1), at the screening dose of 100 mmol/kg, p.o. As
m
shown in Table 3, LASSBio-1298 (5b) and LASSBio-1300 (5e) were
the most active compounds, leading to a decrease in temperature
by 1.8 ꢀC. The compounds LASSBio-1296 (5f), LASSBio-1297 (5c)
and paracetamol (1), in the same conditions, also induced a signif-
icant decrease of body temperature.
m
m
m
heat induces a cutaneous thermonociceptive effect and the stim-
ulus integration occurs due to the stimulation of non-mielinized C
fibers of slow conduction [16]. Although the hot-plate test is
commonly used to assess narcotic analgesics, other centrally acting
drugs, including sedatives and muscle relaxants or psychotomi-
metics, have shown activity in this test while indomethacin and
Aiming to verify the comparative hepatotoxicity of para-
cetamol (1) and its N-phenyl-acetamide sulfonamide analogues
(5a–g), we investigated the effect of paracetamol (1) and
LASSBio-1300 (5e) on liver functions and on the morphology of
liver parenchyma of animals treated for five days with 1 and 5e
(100 mmol/kg, p.o.).
Table 2
Time course effect of derivatives 5a–g (100
m
mol/kg, p.o.), morphine (15
m
mol/kg, i.p.) and paracetamol (100
m
mol/kg, p.o.) in the hot-plate test in mice.
90 min
Substance
n
Latency before the treatmenta (s)
0 min
Time after the treatmenta,b
30 min
60 min
120 min
Control
Morphine
1 Paracetamol
5a (LASSBio-1301)
5b (LASSBio-1298)
5c (LASSBio-1297)
5d (LASSBio-1299)
5e (LASSBio-1300)
5f (LASSBio-1296)
5g (LASSBio-1295)
8
8
8
6
6
6
6
6
6
6
1.8 ꢁ 0.3
1.9 ꢁ 0.2
3.9 ꢁ 0.4
4.7 ꢁ 0.6
4.0 ꢁ 0.9
3.6 ꢁ 0.4
3.9 ꢁ 0.6
3.7 ꢁ 0.6
4.6 ꢁ 0.2
3.0 ꢁ 0.2
3.4 ꢁ 0.8 (47.1%)
9.0 ꢁ 1.4 (78.9%)**
8.1 ꢁ 0.3 (51.8%)
6.1 ꢁ 0.8 (23.0%)
4.1 ꢁ 1.0 (2.4%)
2.1 ꢁ 0.5 (14.3%)
7.5 ꢁ 0.5 (74.7%)**
9.9 ꢁ 2.4* (60.6%)
5.0 ꢁ 0.8 (6.0%)
2.8 ꢁ 0.5 (0%)
3.9 ꢁ 0.8 (53.8%)
4.5 ꢁ 0.5 (57.8%)
8.6 ꢁ 1.7 (54.6%)
7.0 ꢁ 1.0 (32.8%)
4.0 ꢁ 0.5 (0%)
3.4 ꢁ 0.7 (47.1%)
2.8 ꢁ 0.2 (32.1%)
8.6 ꢁ 1.1 (54.6%)
6.2 ꢁ 1.1 (24.2%)
3.4 ꢁ 0.6 (0%)
4.9 ꢁ 0.9 (26.5%)
5.1 ꢁ 1.3 (23.5%)
8.7 ꢁ 1.1** (57.5%)
5.9 ꢁ 0.6 (22.0%)
4.0 ꢁ 0.6 (25.0%)
4.2 ꢁ 0.8 (14.3%)
4.2 ꢁ 0.6 (7.2%)
7.1 ꢁ 1.0* (47.9%)
6.0 ꢁ 0.8 (23.3%)
4.2 ꢁ 0.9 (28.6%)
4.4 ꢁ 0.4 (18.2%)
6.2 ꢁ 0.6 (37.1%)
4.6 ꢁ 0.9 (19.6%)
5.8 ꢁ 0.5 (20.7%)
5.8 ꢁ 0.6 (48.3%)
5.2 ꢁ 0.7 (30.7%)
3.7 ꢁ 0.5 (0%)
4.7 ꢁ 1.0 (21.3%)
5.0 ꢁ 1.0 (8.0%)
8.6 ꢁ 1.4** (65.1%)
a
The readings represent the mean ꢁ S.E.M.
b
The asterisks denote the significance levels in comparison with control groups, **P < 0.01.

M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
3615
200
175
150
125
100
75
Control
10.0
*
Paracetamol
5e (LASSBio-1300)
7.5
**
**
5.0
2.5
0.0
*
50
*
**
25
0
1
Phase
2
ndPhase
st
Fig. 3. Effect of LASSBio-1300 (5e) and paracetamol (1) treatment on serum
measured at 5 days of administration daily (100
U/L (n ¼ 6). Treatment vs. control group: *P < 0.01.
g
GT levels
m
mol/kg, p.o.). Values are expressed in
[100 μmol/kg, p.o.]
Control
5d (LASSBio-1299)
5e (LASSBio-1300)
5f (LASSBio-1296)
5g (LASSBio-1295)
Paracetamol
In attempts to correlate the biological results with the molecular
conformational and stereoelectronic properties, the conformer
distribution of paracetamol (1) and its analogues (5a–g) was
carried out using the semi empirical AM1 Hamiltonian within
Spartan’08 for Windows Software [29,30]. In this study were found
3, 20, 50, 11, 13, 44, 14 and 11 conformers for compounds 1, 5a, 5b,
5c, 5d, 5e, 5f and 5g, respectively. The difference in energy between
the conformer of maximum and minimum energy for all the
studied compounds varied between 6.59 kcal/mol and 11.79 kcal/
mol. The alignment of all the conformers of each compound was
performed based on a selection of a set of atoms (CO, NH, C2, C3, C5,
C6 and SO2) and the results are depicted in Fig. 5. Considering that
a drug’s bioactive conformation often does not correspond to the
absolute minimum of energy, for each set of conformers the
molecular electrostatic potential surface was calculated (Fig. 5).
Others molecular properties, including area, volume, dipole
moment, polar surface area and partition coefficient were calcu-
lated, as available in the Spartan package, and the results obtained
for the conformer of minimum and maximum energy are depicted
in Table 4.
5a (LASSBio-1301)
5b (LASSBio-1298)
5c (LASSBio-1297)
Fig. 2. Effects of paracetamol (1), 5a (LASSBio-1301), 5b (LASSBio-1298), 5c (LASSBio-
1297), 5d (LASSBio-1299), 5e (LASSBio-1300), 5f (LASSBio-1296), 5g (LASSBio-1295) on
the formalin-induced nociception in mice. Animals were treated with paracetamol and
compounds 5a–g (dose ¼ 100
m
mol/kg, p.o.) or the same volume of vehicle (arabic
gum) (the control) 40 min before 20
m
L of 2% formalin in 0.9% saline was injected s.c.
into the dorsal surface of the right hind paw. The time spent licking and biting the
injected paw was measured with a hand-stop watch from 0 to 5 min (the first pha-
sedA) and from 15 to 30 min (the second phasedB) after formalin injection. Data
were presented as the mean ꢁ S.E.M. (n ¼ 6). *P < 0.05, **P < 0.01 vs. control.
For the liver function the serum gamma-Glutamyl Transferase
(gGT) activity was determined. The gGT is an enzyme embedded in
the hepatocyte plasma membrane and considered to be one of the
best indicators of liver damage [28]. As illustrated in Fig. 3, mice
treated with paracetamol (1) showed a significant increase in
serum gGT, when compared to control, while no significant effect
was observed for LASSBio-1300 (5e).
As seen in Fig. 5, two main differences can be drawn
comparing the set of conformers of paracetamol (1) and its
analogues. These differences are based on the increase in area
and volume, introduced by the replacement of the phenolic
hydroxyl group by a sulfonamide unit, and the introduction of
a new negative potential region, localized at the terminal moiety
of piperazine, N-methyl-piperazine, morpholine and thio-
morpholine rings, present in the structures of compounds 5d, 5e,
5f and 5g, respectively. Considering the better peripheral anal-
gesic activity for paracetamol analogues, with exception of
compound 5c, (Table 1) we can speculate that the improvement
on the electron-rich regions, represented by the eletrostatic
The microscopic liver analysis of animals treated with saline
(control group) and with LASSBio-1300 (5e) demonstrated
a regular morphology of the liver parenchyma, with well-designed
and evident hepatic cells and sinusoids (Fig. 4A and B). On the other
hand, the liver of animals treated with paracetamol (1) showed
histological changes in the micro architecture of the liver lobule
and degenerated hepatocytes, showing perinuclear vacuolization in
most areas (Fig. 4C).
Taken together, these results seem to demonstrate hepatic cell
damage caused by paracetamol (1), which has not been observed in
the treatment with LASSBio-1300 (5e) in the same conditions.
Table 3
Activity of derivatives 5a–g (100 mmol/kg, p.o.) and paracetamol (100 mmol/kg, p.o.) on the body temperature of mice.
Substance
n
Temperature (ꢀC) (mean ꢁ S.E.M) (before treatment)a
Temperature (ꢀC) (mean ꢁ S.E.M) (after treatment)a
D
Temperatureb (ꢀC)
1 Paracetamol
8
8
8
8
8
8
8
8
38.0 ꢁ 0.2
36.4 ꢁ 0.3
37.8 ꢁ 0.2
37.3 ꢁ 0.2
36.6 ꢁ 0.4
37.3 ꢁ 0.4
37.8 ꢁ 0.1
37.5 ꢁ 0.2
36.9 ꢁ 0.3
36.6 ꢁ 0.2
36.0 ꢁ 0.3
36.5 ꢁ 0.2
36.8 ꢁ 0.3
35.5 ꢁ 0.2
36.4 ꢁ 0.2
36.8 ꢁ 0.2
ꢂ1.1**
0.2
ꢂ1.8**
ꢂ0.8*
0.2
ꢂ1.8**
ꢂ1.4**
ꢂ0.7
5a (LASSBio-1301)
5b (LASSBio-1298)
5c (LASSBio-1297)
5d (LASSBio-1299)
5e (LASSBio-1300)
5f (LASSBio-1296)
5g (LASSBio-1295)
a
The readings represent the mean ꢁ S.E.M. of 8 animals.
b
The asterisks denote the significance levels in comparison with control groups, **P < 0.01.

3616
M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
Fig. 4. Histological observation of the liver. Mice were treated with LASSBio-1300 (5e, 100 mmol/kg, p.o.) or paracetamol (100 mmol/kg, p.o.) for 5 days. Control group was
administered only with vehicle. Hepatic tissue was collected 24 h after the last treatment and subjected to H&E staining. Left slides are 100ꢄ magnification and right slides are 400ꢄ
magnification. The arrow in the figure indicates the hepatocyte necrosis or hepatocyte degeneration.
potential surfaces illustrated in Fig. 5, was beneficial to the
analgesic profile of compounds 5a and 5b and 5d–g. Although,
the more active compound (5e, LASSBio-1300) being the one
with higher volume, area and dipole moment, none direct
correlation between the calculated stereoelectronic properties
and the biological results can be drawn. This fact can be
explained considering the limitation of try to establish a struc-
ture–activity relationship when the target/mechanism of action is
unknown, and also when the pharmacological profile was
determined in vivo, the results of which are, consequently,
influenced by pharmacokinetic parameters.
Aiming to establish the theoretical potential to qualify
compounds 5a–g as drug candidates, we calculated the overall
drug-score values, using a web-based system named Osiris Prop-
erty
Explorer
(http://www.organic-chemistry.org/prog/peo/)
which combines drug-likeness, solubility, c log P, molecular weight,
and toxicity risks in one handy value that may be used to judge the
compound’s overall potential to qualify for a drug. Values ¼ 1.0
mean no risk and values ꢃ0.6 mean high risk [31]. As illustrated in
Table 4, the N-phenyl-acetamide sulfonamide derivatives (5a–g)
presented a higher drug-score than prototype paracetamol, with
the exception of compound 5b.

M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
3617
Fig. 5. Three-dimensional isopotential surface of all the conformers of paracetamol (1) and its analogues 5a–g. The solid contours represent a constant potential of ꢂ83.68 kJ/mol
showing the position and orientation of the lone pair electrons at approximately 1.4 Å away from the van der Waals surface of the molecule.
3. Conclusions
(ID50 ¼ 19.27
m
mol/kg, p.o.) and presenting a better hypothermic
profile in a screening dose of 100
mmol/kg. The increase of area,
A new series of analgesic drug candidates were identified. In
this series (5a–g), planned by molecular modifications on the
prototype paracetamol (1), compound LASSBio-1300 (5e;
volume and eletrostatic potential of paracetamol’s analogues
seems to be beneficial for the analgesic activity. Unlike para-
cetamol (1) and the other analogues (5a, 5d–g), compounds 5b
and 5c presented an important anti-hypernociceptive activity
associated to inflammatory pain.
ID50 ¼ 5.81
mmol/kg, p.o.) stands out as a new non-hepatotoxic
drug candidate, more potent than the standard
1
Chart 1.

3618
M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
Table 4
Selected molecular properties: heat of formation (
and Drug-score values.
DHf), surface area, volume of a space-filling model, dipole, polar surface area (PSA), calculated partition coefficient (c log P)
Compound
D
Hfa (kcal/mol)
Areaa (Å2)
Volumea (Å3)
Dipolea (Debye)
PSAa (Å2)
c log Pb
Drug-scorec
0.20
1
ꢂ247.30
ꢂ237.35
ꢂ371.22
ꢂ363.91
ꢂ369.13
ꢂ359.88
ꢂ379.37
ꢂ372.78
ꢂ300.00
ꢂ291.78
ꢂ288.19
ꢂ276.40
ꢂ497.15
ꢂ488.37
ꢂ313.88
ꢂ306.33
180.99
178.96
265.72
263.11
300.66
299.71
301.60
299.63
297.84
295.96
316.71
315.97
293.93
292.28
301.65
299.53
158.47
157.85
232.41
231.59
270.91
270.40
275.89
275.21
270.45
269.77
289.83
289.23
267.35
266.68
276.01
275.28
2.61
2.48
7.04
4.31
7.0
4.54
6.80
4.89
5.97
4.66
7.58
4.79
6.70
3.53
6.63
3.39
42.86
45.07
67.60
69.86
55.32
57.71
55.19
58.03
67.73
58.03
57.65
60.61
63.05
65.82
55.17
57.78
0.494
5a
5b
5c
5d
5e
5f
0.791
1.874
2.009
0.748
1.323
0.762
1.497
0.95
0.56
0.91
0.95
0.95
0.95
0.93
5g
a
Calculated using Spartan’08 for Windows.
Calculated using ChemOffice 2004 (Chem3D Ultra 8.0) Software.
Calculated using Osiris program (http://www.organic-chemistry.org/prog/peo/).
b
c
4. Experimental section
4.1.2. 4-(Acetylamine)-benzenesulfonyl chloride (7)
9.12 g (5.2 mL, 78.27 mmol) of chlorosulfonic acid was slowly
added to 2.0 g (14.80 mmol) of N-phenyl-acetamide (3). The
resulting mixture was stirred and heated at 60 ꢀC for 30 min. After
cooling, the 4-(acetylamine)-benzenesulfonyl chloride (7) was
filtered through a Buckner funnel, washed twice with 100 mL of
water and recovered as a white hygroscopic powder in 85% yield,
mp 143–148 ꢀC. The melting point and IR data for compound (7) are
in agreement with previews reports [8–10].
4.1. Chemistry
Reactions were routinely monitored by thin-layer chromatog-
raphy (TLC) in silica gel (F245 Merck plates) and the products
visualized with iodine or ultraviolet lamp (254 and 365 nm). 1H and
13C nuclear magnetic resonance (NMR) spectra were determined in
DMSO-d6 solutions using a Bruker AC-200 spectrometer. Peak
positions are given in parts per million (
d) from tetramethylsilane
IR (nmax, KBr) n
(cmꢂ1): 3310, 1683, 1374, 1168, 839.
as internal standard, and coupling constant values (J) are given in
Hz. Signal multiplicities are represented by: s (singlet), d (doublet),
t (triplet), q (quadruplet), qu (quintuplet) and m (multiplet).
Infrared (IR) spectra were obtained using an ABB FTLA2000-100 IR
spectrometer. Samples were examined as potassium bromide (KBr)
disks. Elemental microanalyses were obtained on an Elemental
Analyzer (Flash EA 1112 Series, Thermo Scientific) from vacuum-
dried samples. The analytical results for C, H, and N were within
ꢁ0.4% of the theoretical values. Melting points were determined
using a Quimis instrument and are uncorrected. All described
products showed 1H and 13C NMR spectra according to the assigned
structures. All organic solutions were dried over anhydrous sodium
sulphate and all organic solvents were removed under reduced
pressure in rotatory evaporator.
4.1.3. General procedures for the preparation of sulfonamides 5a–g
The functionalized amines (5.0 mmol) were added to
a solution of sulfonyl chloride derivative (7) (0.5 g; 2.14 mmol)
in 50 mL of methylene chloride. The reaction mixture was
stirred for about 30 min, at room temperature, when the end of
the reaction was observed by TLC. The sulfonamide derivatives
5a–g were isolated by addition of 50 mL of methylene chloride
and extraction with 10% aq HCl and brine. The organic layer
was dried over anhydrous Na2SO4 and evaporated at reduced
pressure to give the sulfonamide derivatives 5a–g in good
yields.
4.1.3.1. N-[4-(Ethylamino-4-ylsulfonyl)phenyl]acetamide (5a; LASS-
Bio-1301). The title compound was obtained as a white crystal, in
50% yield, by condensing 7 with ethylamine, mp 155–157 ꢀC.
4.1.1. N-Phenyl-acetamide (3)
A solution of 2.1 g (25.6 mmol) of sodium acetate anhydrous in
8.4 g (8.0 mL,139.9 mmol) of glacial acetic acid was prepared. 8.18 g
of aniline (8.0 mL, 87.8 mmol) was slowly added, and then 9.2 g
(8.5 mL, 90.1 mmol) of acetic anhydride was also added. The reac-
tion mixture was stirred for about 30 min, at room temperature,
when the end of reaction was observed by TLC. After cooling, the N-
phenyl-acetamide (3) was filtered through a Buckner funnel,
washed twice with 200 mL of water and recovered as a white shiny
powder in 90% yield, mp 116 ꢀC.
1H NMR (200 MHz, DMSO-d6, TMS)
d (ppm): 0.94 (t, 3H,
J ¼ 7.2 Hz, RNHCH2CH3); 2.08 (s, 3H, COCH3); 2.74 (qu, 2H, J ¼ 7.2 Hz
and 5.7 Hz, RNHCH2CH3); 7.42 (t, 1H, J ¼ 5.7 Hz, RNHCH2CH3); 7.69
(d, 2H, J ¼ 8.9 Hz, H2 and H6); 7.77 (d, 2H, J ¼ 8.9 Hz, H3 and H5);
10.41 (s, 1H, CONH).
13C NMR (50 MHz, DMSO-d6, TMS)
d (ppm): 14.7 (RNHCH2CH3);
24.3 (COCH3); 37.5 (RNHCH2CH3); 118.7 (C-2 and C-6); 127.6 (C-3
and C-5); 134.2 (C-4); 142.7 (C-1); 169.1 (C]O).
IR (nmax, KBr)
n
(cmꢂ1): 3330, 3149, 1679, 1318, 1154, 834.
1H NMR (200 MHz, DMSO-d6, TMS)
d
(ppm): 2.03 (s, 3H,
Anal. Calcd. for C10H14N2O3S: C, 49.57; H, 5.82; N, 11.56. Found:
C, 49.29; H, 5.66; N, 11.45.
COCH3); 7.01 (t,1H, J ¼ 7.5 Hz, H4); 7.28 (t, 2H, J ¼ 8.0, 7.5 Hz, H3 and
H5); 7.57 (d, 2H, J ¼ 8.0 Hz, H2 and H6); 9.93 (s, 1H, CONH).
13C NMR (50 MHz, DMSO-d6, TMS)
d
(ppm): 24.6 (COCH3); 119.5
4.1.3.2. N-[4-(Diethylamino-4-ylsulfonyl)phenyl]acetamide (5b;
LASSBio-1298). The title compound was obtained as an orange
solid, in 59% yield, by condensing 7 with diethylamine, mp
71–73 ꢀC.
(C-3 and C-5); 123.5 (C-4); 129.2 (C-2 and C-6); 139.9 (C-1); 168.8
(C]O).
IR (nmax, KBr)
n
(cmꢂ1): 3299, 1666, 755, 698.
Anal. Calcd. for C8H9NO: C, 71.09; H, 6.71; N, 10.36. Found: C,
71.02; H, 6.78; N, 10.38.
1H NMR (200 MHz, DMSO-d6, TMS)
d
(ppm): 1.01 (t, 6H,
J ¼ 7.2 Hz, RN(CH2CH3)2); 2.08 (s, 3H, COCH3); 3.12 (q, 4H, J ¼ 7.2 Hz,

M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
3619
RN(CH2CH3)2); 7.70 (d, 2H, J ¼ 9.0 Hz, H2 and H6); 7.77 (d, 2H,
J ¼ 9.0 Hz, H3 and H5); 10.34 (s, 1H, CONH).
4.1.3.7. N-[4-(Thiomorpholin-4-ylsulfonyl)phenyl]acetamide (5g; LA-
SSBio-1295). The title compound was obtained as a yellow powder,
in 65% yield, by condensing 7 with thiomorpholine, mp 207–
209 ꢀC.
13C NMR (50 MHz, DMSO-d6, TMS)
d (ppm): 14.1 (RN(CH2CH3)2);
24.2 (COCH3); 41.8 (RN(CH2CH3)2); 118.8 (C-2 and C-6); 127.9 (C-3
and C-5); 133.4 (C-4); 143.0 (C-1);169.1 (C]O).
1H NMR (200 MHz, DMSO-d6, TMS)
d (ppm): 2.09 (s, 3H,
IR (nmax, KBr)
n
(cmꢂ1): 3235, 1681, 1330, 1149, 935, 848.
COCH3); 2.65 (t, 4H, J ¼ 5.0 Hz, H20 and H60); 3.16 (t, 4H, J ¼ 5.0 Hz,
H30 and H50); 7.67 (d, 2H, J ¼ 8.9 Hz, H2 and H6); 7.82 (d, 2H,
J ¼ 8.9 Hz, H3 and H5); 10.40 (s, 1H, CONH).
Anal. Calcd. for C12H18N2O3S: C, 53.31; H, 6.71; N, 10.36. Found:
C, 53.22; H, 6.78; N, 10.28.
13C NMR (50 MHz, DMSO-d6, TMS)
d (ppm): 24.2 (COCH3); 26.4
4.1.3.3. N-[4-(Piperidin-4-ylsulfonyl)phenyl]acetamide (5c; LASSBio-
1297). The title compound was obtained as a yellow crystal, in 70%
yield, by condensing 7 with piperidine, mp 118–120 ꢀC.
(C-30 and C-50); 47.8 (C-20 and C-60); 118.8 (C-2 and C-6); 128.5 (C-3
and C-3); 129.5 (C-4); 143.5 (C-1); 169.2 (C]O).
IR (nmax, KBr)
n
(cmꢂ1): 3352, 1702, 1321, 1153, 835.
1H NMR (200 MHz, DMSO-d6, TMS)
d
(ppm): 1.34 (m, 2H, H40);
Anal. Calcd. for C12H16N2O3S2: C, 47.98; H, 5.37; N, 9.33. Found:
C, 47.84; H, 5.33; N, 9.24.
1.52 (m, 4H, H30 and H50); 2.09 (s, 3H, COCH3); 2.83 (t, 4H,
J ¼ 5.3 Hz, H20 and H60); 7.64 (d, 2H, J ¼ 8.9 Hz, H2 and H6); 7.80 (d,
2H, J ¼ 8.9 Hz, H3 and H5); 10.38 (s, 1H, CONH).
13C NMR (50 MHz, DMSO-d6, TMS)
d
(ppm): 23.5 (COCH3); 24.7
4.2. Biological assays
(C-40); 25.2 (C-30 and C-50); 47.2 (C-20 and C-60); 119.2 (C-2 and C-6);
129.2 (C-3 and C-5); 129.5 (C-4); 143.9 (C-1);169.7 (C]O).
4.2.1. Animals
IR (nmax, KBr)
n
(cmꢂ1): 3261, 1675, 1336, 1162, 850.
Swiss mice weighing 20–30 g (from the BIOCEN-UFAL) were
housed in group cages and maintained on a 12 h light/12 h dark
cycle. Animals had free access to food and water at all times.
Experiments were carried out according to a protocol approved by
the Animal Welfare Committee of Federal University of Alagoas
(UFAL) and in according with the ethical guidelines for investiga-
tion of experimental pain in conscious animals [33].
Anal. Calcd. for C13H18N2O3S: C, 55.30; H, 6.43; N, 9.92. Found: C,
55.24; H, 6.47; N, 9.72.
4.1.3.4. N-[4-(Piperazin-4-ylsulfonyl)phenyl]acetamide (5d; LASSBio-
1299). The title compound was obtained as a beige powder, in 50%
yield, by condensing 7 with piperazine, mp 150–152 ꢀC.
1H NMR (200 MHz, DMSO-d6, TMS)
d (ppm): 2.09 (s, 3H,
COCH3); 2.39 (br, 1H, NH); 2.73 (s, 8H, H20-H60); 7.64 (d, 2H,
J ¼ 8.9 Hz, H2 and H6); 7.82 (d, 2H, J ¼ 8.9 Hz, H3 and H5); 10.41 (s,
1H, CONH).
4.2.2. Reagents
Acetic acid (Merck), arabic gum (Sigma Aldrich), morphine
sulphate (Dimorf-Cristalia-BR), and paracetamol (Sigma Aldrich)
were obtained from commercial sources. A solution of formalin
2.5% was prepared with formaldehyde (Merck) in saline (NaCl 0.9%).
Paracetamol (1) and its analogues 5a–g were used as suspension in
arabic gum in all the experiments and oral administrations.
13C NMR (50 MHz, DMSO-d6, TMS)
d (ppm): 24.7 (COCH3); 45.1
(C-30 and C-50); 47.1 (C-20 and C-60); 119.2 (C-2 and C-6); 128.8 (C-
4); 129.4 (C-3 and C-5); 144.0 (C-1); 169.7 (C]O).
IR (nmax, KBr)
n
(cmꢂ1): 3410, 3294, 1687, 1342, 1172, 840.
Anal. Calcd. for C12H17N3O3S: C, 50.87; H, 6.05; N, 14.83. Found:
C, 50.77; H, 6.08; N, 14.87.
4.2.3. Acetic acid-induced writhing
This test was performed as described by Collier et al. [12],
Koster et al. [13] and Fontenele et al. [14]. Acetic acid (0.6%, v/v)
was administered i.p. in a volume of 0.1 mL/10 g. The number of
writhes, a response consisting of contraction of an abdominal
wall, pelvic rotation followed by hind limb extension, was
counted during continuous observation for 20 min beginning
from 5 min after the acetic acid injection. Paracetamol (1) and
the N-phenyl-acetamide sulfonamide derivatives (5a–g) (all
4.1.3.5. N-[4-[(4-Methyl-1-piperazinyl)sulfonyl]phenyl]acetamide (5e;
LASSBio-1300). The title compound was obtained as a yellow crystal,
in 67% yield, by condensing 7 with N-methyl-piperazine, mp 175 ꢀC.
1H NMR (200 MHz, DMSO-d6, TMS)
d (ppm): 2.09 (s, 3H,
COCH3); 2.12 (s, 3H, RNCH3); 2.33 (t, 4H, J ¼ 4.8 Hz, H30 and
H50); 2.84 (t, 4H, J ¼ 4.8 Hz, H20 and H-60); 7.65 (d, 2H, J ¼ 8.5 Hz,
H2 and H6); 7.82 (d, 2H, J ¼ 8.5 Hz, H3 and H5); 10.41 (s, 1H,
CONH).
100
mmol/kg, oral administration) were administered 60 min
13C NMR (50 MHz, DMSO-d6, TMS)
d
(ppm): 24.2 (COCH3); 45.3
before the acetic acid injection. Antinociceptive activity was
expressed as inhibition percent of the usual number of writhing
observed in control animals. Dose–response curves were
obtained for paracetamol (1) and LASSBio-1300 (5e) (100, 30, 10
(RNCH3); 45.8 (C-30 and C-50); 53.5 (C-20 and C-60); 118.7 (C-2 and
C-6); 128.3 (C-4); 128.8 (C-3 and C-5); 143.5 (C-1); 169.2 (C]O).
IR (nmax, KBr)
n
(cmꢂ1): 3345, 1704, 1343, 1162, 839.
Anal. Calcd. for C13H19N3O3S: C, 52.51; H, 6.44; N, 14.13. Found:
C, 52.34; H, 6.59; N, 14.03.
and 3 mmol/kg) using groups of 8 animals. Control animals
received the vehicle. The ID50 values (i.e. dose which reduces
response by 50% relative to the control values), to paracetamol
and LSSBio-1300 (5e) were determined by linear regression from
individual experiments with linear regression Graph Pad Prisma
softwareÒ.
4.1.3.6. N-[4-(Morpholin-4-ylsulfonyl)phenyl]acetamide (5f; LASS-
Bio-1296). The title compound was obtained as a yellow crystal, in
55% yield, by condensing 7 with morpholine, mp 174–176 ꢀC.
1H NMR (200 MHz, DMSO-d6, TMS)
d (ppm): 2.09 (s, 3H,
COCH3); 2.81 (t, 4H, J ¼ 4.8 Hz, 4.3 Hz, H20 and H60); 3.61 (t, 4H,
J ¼ 4.8 Hz, 4.3 Hz, H30 and H50); 7.65 (d, 2H, J ¼ 8.9 Hz, H2 and H6);
7.83 (d, 2H, J ¼ 8.9 Hz, H3 and H5); 10.41 (s, 1H, CONH).
4.2.4. Hot-plate test
Mice were treated according to the method described by Fran-
zotti et al. [15]. The animals (n ¼ 6) were placed on a hot-plate set at
55 ꢁ1 ꢀC. Reaction time was recorded when the animals licked their
fore and hind-paws and jumped at 30, 60, 90 and 120 min after oral
13C NMR (50 MHz, DMSO-d6, TMS)
d (ppm): 24.2 (COCH3); 45.9
(C-20 and C-60); 65.3(C-30 and C-50); 118.7 (C-2 and C-6); 127.8 (C-
4); 129.0 (C-3 and C-5); 143.7 (C-1); 169.2 (C]O).
administration of 100
acetamide sulfonamide derivatives (5a–g) or reference drug
(morphine,15 mol/kg. i.p.). Baseline was considered as the mean of
reaction time obtained at 30 and 60 min before administration of
mmol/kg of paracetamol (1) or the N-phenyl-
IR (nmax, KBr)
n
(cmꢂ1): 3305, 1679, 1344, 830.
Anal. Calcd. for C12H16N2O4S: C, 50.69; H, 5.67; N, 9.85. Found: C,
50.67; H, 5.67; N, 9.76.
m

3620
M.L. de Castro Barbosa et al. / European Journal of Medicinal Chemistry 44 (2009) 3612–3620
5a–g or 1 or morphine and was defined as normal reaction of animal
to the temperature.
semi empirical AM1 Hamiltonian [29] within Spartan’08 Windows
program on a Pentium IV 1.5 GHz. The stereoelectronic properties
were calculated on all the conformers of the molecules, as available
in the Spartan package [30].
4.2.5. Formalin-induced nociception
The procedure used was essentially the same as that described
previously [20,21]. Animals received 20 mL of 2.5% formalin solution
Acknowledgements
(0.92% formaldehyde in saline) in the ventral surface of the right
hind paw. Animals were observed from 0 to 5 min (neurogenic
phase) and from 15 to 30 min (inflammatory phase) and the time
that they spent licking the injected paw was recorded and
considered as indicative of nociception. Animals received para-
cetamol (1) or the N-phenyl-acetamide sulfonamide derivatives
The authors thank CAPES (BR), CNPq (BR), FAPEAL (BR), FAPERJ
(BR) and IM-INOFAR (BR, #420015/05-1) for fellowship and finan-
cial support.
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4.2.7. Serum enzyme assessment
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mmol/kg,
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m
days. 24 h after the last dose of vehicle, paracetamol and LASSBio-
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