Synthesis, pharmacological evaluation and electrochemical studies of novel 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives: Discovery of LASSBio-881, a new ligand of cannabinoid receptors

Article abstract of DOI:10.1016/j.bmc.2007.01.013

We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to bind to CB1 receptors (71% at 100 μM) and also to inhibit T-cell proliferation (66% at 10 μM) probably by binding to CB2 receptors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an important antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition, we showed that these structural sub-units are essential for the observed pharmacological activity.

Full text of DOI:10.1016/j.bmc.2007.01.013

Bioorganic & Medicinal Chemistry 15 (2007) 2421–2433
Synthesis, pharmacological evaluation and electrochemical studies
of novel 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazone
derivatives: Discovery of LASSBio-881, a new ligand of
cannabinoid receptors
Carolina D. Duarte,a,b Jorge L. M. Tributino,a,c Daniel I. Lacerda,a,c Marina V. Martins,a,c
Magna S. Alexandre-Moreira,d Fernando Dutra,e Etelvino J. H. Bechara,e
Francine S. De-Paula,f Marilia O. F. Goulart,f Juliano Ferreira,g Joao B. Calixto,g
˜
Marise P. Nunes,h Alvaro L. Bertho,h Ana Luisa P. Miranda,a
Eliezer J. Barreiroa,b and Carlos A. M. Fragaa,b,
*
a
ˆ
LASSBio—Laborato´rio de Avaliac¸a˜o e Sı´ntese de Substancias Bioativas, Faculdade de Farmacia,
Universidade Federal do Rio de Janeiro, PO Box 68006, 21944-971, Rio de Janeiro, RJ, Brazil
´
b
ˆ
´
Programa de Po´s-Graduac¸a˜o em Quı´mica Organica, Instituto de Quımica, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, Brazil
c
ˆ
Departamento de Farmacologia Ba´sica e Clı´nica, Instituto de Ciencias Biomedicas,
´
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
LaFI—Laborato´rio de Farmacologia e Imunidade, Departamento de Fisiologia, Centro de Ciencias Biologicas,
d
ˆ
´
Universidade Federal de Alagoas, Maceio´, AL, Brazil
eDepartamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil
fInstituto de Quı´mica e Biotecnologia, Universidade Federal de Alagoas, Maceio´, AL, Brazil
g
ˆ
´
Departamento de Farmacologia, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil
´
hDepartamento de Imunologia, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
Received 3 December 2006; accepted 11 January 2007
Available online 17 January 2007
Abstract—We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro
antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to
bind to CB1 receptors (71% at 100 lM) and also to inhibit T-cell proliferation (66% at 10 lM) probably by binding to CB2 recep-
tors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an impor-
tant antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the
presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition,
we showed that these structural sub-units are essential for the observed pharmacological activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ery of an injured tissue or that could lead to persistent
tissue damage by inflammatory cells if assisted repair
is not properly achieved.1 Some of these mediators, for
example, eicosanoids, are also able to activate and sen-
sitize nociceptors, leading to hyperalgesic response.2
The contribution of the eicosanoids to the genesis of sev-
eral pathological states, such as chronic inflammatory
diseases, thrombosis, and pain, is well established.3
The biosynthesis of such mediators involves a key
enzyme, that is, cyclooxygenase (COX).4 Its constitutive
The inflammatory response is a complex set of interac-
tions between cell-derived mediators to attain the recov-
Keywords: N-Acylhydrazone derivatives; Cannabinoid; Analgesic;
Anti-inflammatory; Antioxidant activities.
*
Corresponding author. Tel.: +55 21 25626503; fax: +55 21
25626644; e-mail: cmfraga@pharma.ufrj.br
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.013

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C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
isoform, named COX-1, is related to homeostatic func-
tions, such as gastric mucosal cytoprotection, renal
blood flow regulation, and vascular antithrombotic
activity; while its inducible isoform, COX-2, is overex-
pressed under stress conditions, although constitutive
in a few tissues.5
In addition, the role of the endocannabinoid system in
the endogenous control of pain6 and inflammation is
widely described in the literature,7 due to the partial
agonism of anandamide (AEA, 1) (Chart 1) and other
endocannabinoids on cannabinoid receptors CB1 and
CB2, expressed mainly in brain and immune cells,
respectively.8 Although the activation of brain CB1
receptors leads to undesirable side effects related to Can-
nabis ingestion, the therapeutic usefulness of CB1 ago-
nists is still valuable in neurotrauma, brain ischemia,
pain, and glaucoma. On the other hand, the absence
of such psychotropic events on the selective agonism
of CB2 receptors has called the attention of medicinal
chemists on the development of novel anti-inflammatory
and analgesic drugs.9 The wide chemical diversity of nat-
ural and synthetic cannabinoid agonists does not permit
the election of a common pharmacophore, since the
structure–activity relationships could not be well
established.9
Scheme 1. Design concept of new 6-nitro-3,4-methylenedioxyphenyl-
N-acylhydrazone derivatives (3).
by the relative acidity of the amide hydrogen of the
NAH group as well as its capacity of stabilizing free
radicals.15
Other enzymes involved in the inflammatory hyperalge-
sic response, that is, COX-2 and lipoxygenases, are also
able to convert AEA in oxygenated products (prosta-
mides), which can bind to cannabinoid receptors,10
showing a close connection between these two pathways
in a way to control the inflammatory process.
In this context, the aim of our present work was the
development of novel drug candidates with an in vivo
anti-inflammatory and antinociceptive profile, present-
ing also an antioxidant activity, based on our previous
results concerning the medicinal chemistry of NAH
derivatives.16 The rational approach leading to the
new 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazones
(3) (Scheme 1) includes the molecular hybridization17
As a consequence of inflammatory response, there is a
massive production of reactive oxygen species (ROS),
which have been recently recognized as true messengers,
and even their toxic effects are viewed as the result of the
perversion of an otherwise physiological extra/intracel-
lular signaling.11 This is crucial in inflammatory disor-
ders in the central nervous system, for example,
cerebral ischemia-reperfusion injury, Alzheimer’s dis-
ease, and pre-senile dementia.12 Nimesulide (2) was
recently described as neuroprotective both in vivo and
in vitro, and this activity is due to both anti-inflammato-
ry and antioxidant properties.13
between LASSBio-294 (4),
a
potent analgesic
(ID50 = 8.1 lmol/kg—abdominal constrictions induced
by acetic acid in mice) and anti-platelet derivative
(IC50 = 15.3 lM—rabbit platelet aggregation induced
by AA, inhibiting the production of thromboxane B2)
but poor anti-inflammatory,18 and nimesulide (2), an
anti-inflammatory drug with in vivo antioxidant proper-
ties,13 in order to optimize the anti-inflammatory profile
of lead-compound (4). The nature of the aryl groups
attached to the imine sub-unit of compounds (3)
that are 2-thiophenyl, 2-furyl, 2-pyrrolyl, 2-pyridinyl,
4-pyridinyl, and phenyl was selected based on classical
ring isosteric replacement.17 In addition, we elected the
3,5-di-tert-butyl-4-hydroxyphenyl group in an attempt
to optimize the radical scavenging potential.19 We also
synthesized compound (5) (Scheme 1), to evaluate the
possible contribution of the nitro group in the modula-
tion of the redox properties and bioactivity.
N-Acylhydrazones (NAH) have been widely described
by our group as potent anti-inflammatory, antinocicep-
tive, and anti-platelet compounds,14 due to their ability
to mimic the bis-allylic moiety of unsaturated fatty acids
and amides, for example, arachidonic acid (AA), precur-
sor of eicosanoid biosynthesis, and AEA (1), involved in
the endocannabinoid system.15 This can be rationalized
2. Results and discussion
2.1. Chemistry
O
OH
N
H
The new substituted 6-nitro-3,4-methylenedioxyphenyl-
N-acylhydrazone derivatives (3) and (5) were synthe-
sized from safrole (6) (Scheme 2), an abundant natural
product from Brazilian flora, the major constituent of
CH
3
Chart 1.

C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
2423
Scheme 2. Synthesis of compounds (3a–g and 5). Reagents and conditions: (a) KOHaq 3 N, n-BuOH, rt, 3h; (b) i—O3/O2, AcOH, 0 ꢁC, l h; ii—Znꢁ,
AcOH (75%, three steps); (c) HNO3, 20–25 ꢁC, 0.5 h, 95%; (d) I2, KOH, MeOH, 0 ꢁC, 1.5 h, 88%; (e) N2H4ÆH2O 80%, EtOH, ta, l h, 70%; (f) ArCHO,
EtOH, HCIcat, rt, 0.5 h.
Sassafras oil from Ocotea pretiosa and of extracts from
Piper hispidinervum20 (Scheme 1). After isomerization
and oxidative cleavage,21 we obtained piperonal (7) in
75% yield from (6), which was submitted to nitration22
with concentrated nitric acid at 20–25 ꢁC, and further
oxidation14c to the corresponding methyl ester (9) by
treatment of the aldehyde (8) with 2.6 equiv of KOH
and 1.3 equiv of iodine in methanol at 0 ꢁC, in 94% yield
over two steps. Absorption bands at 1509 and
1038 cmꢀ1 in the infrared spectrum clearly show the
presence of the nitro group; additionally, the presence
of two singlets at 7.03 and 7.38 ppm in the aromatic
region of the H NMR spectrum characterizes the AB
pattern of the aromatic ring. These data, which are in
complete agreement with previous results from the liter-
ature,22 demonstrate unequivocally that the nitration
occurred regioselectively at the position 6 of the 1,3-ben-
zodioxole ring. The key hydrazide intermediate (10) was
then obtained in 70% yield, by treating an ethanolic
solution of (9) with hydrazine hydrate at room
1
Table 1. New NAH target compounds (3a–g) and (5)
Compound
Substituent
Yield (%)
88
Melting point (ꢁC)
E/Z ratioa
O
3a LASSBio-879
226–228
60:40
S
3b LASSBio-880
3c LASSBio-881
90
82
240–242
280–282
60:40
67:33
OH
3d LASSBio-882
3e LASSBio-934
3f LASSBio-935
3g LASSBio-936
95
84
89
83
216–218
184–185
202–203
212–213
70:30
70:30
70:30
57:43
N
N
H
N
5 LASSBio-945
91
231–232
—
OH
a Calculated from 1H NMR iminic hydrogen integration, according to Karabatsos et al.23

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C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
temperature.14c Piperonylhydrazide (10a) was also ob-
tained from safrole (6), using the same synthetic strate-
gy, as previously described by our group.14c
36.4%, respectively, without provoking gastric irritation
(data not shown). On the other hand, 2-pyridinyl deriv-
ative LASSBio-934 (3e) presented a moderate ability to
inhibit the edema formation, that is, 22.8%, mimicking
the anti-inflammatory profile of prototype LASSBio-
294 (4).18
Finally, the new NAH target compounds (3a–g) and
(5) (Table 1) were obtained, in 70–95% yield, by
acid-catalyzed condensation of (10) with the appropriate
aromatic aldehydes in ethanol.14c Our next step was to
determine the relative configuration of the imino double
bond in NAH derivatives (3a–g), in order to assure the
Thus, LASSBio-881 (3c) and LASSBio-945 (5) were
selected for further evaluation of their anti-inflammato-
ry activity in the phorbol ester (TPA)- and AA-induced
murine ear edema assays.26 The first model is character-
ized by an important participation of the 5-lipoxygenase
(5-LO) pathway products, and COX-2 inhibitors do not
have any activity in this assay, although they are able to
reduce the AA-induced ear edema. The results depicted
in Table 3 show that only LASSBio-881 (3c) was able to
inhibit in 36% the AA-induced ear edema, however, it is
not statistically significant even showing a pharmacologic
importance.
1
diastereomeric ratio. Analysis of the H NMR spectra
revealed the presence of two singlet signals in an average
proportion of 65:35, corresponding to the E and Z
imine-attached hydrogens, respectively (Table 1), due
to the stereoelectronic effects of the nitro group ortho
to the NAH moiety. The assignment of each diastereo-
mer was made in agreement with the previous work of
Karabatsos et al.23 for the relative configuration of
hydrazones and related compounds, which describes
that the imino hydrogen of the E-diastereomer is
0.2–0.3 ppm downfield from the corresponding Z-dia-
stereomer.23 On the other hand, we detected the forma-
tion of only one diastereomer of compound (5), which
corresponds to the E configuration, according to previ-
ous results with LASSBio-294 (4) and analogs.24
LASSBio-881 (3c) and LASSBio-945 (5) were unable to
inhibit either phorbol ester or AA-induced edema, as
well as LASSBio-294 (4).18 These results indicate that
these compounds do not have an inhibitory effect at
the AA-cascade level.
2.2. Anti-inflammatory and analgesic activities
Besides the anti-inflammatory activity of compounds
LASSBio-881 (3c) and LASSBio-945 (5), we have
also investigated their antinociceptive profile in the
formalin-induced murine hypernociception model27
(Table 4). This assay elicits a neurogenic phase followed
by an inflammatory event. In contrast to nimesulide (2),
which was able to prevent inflammatory nociception,
LASSBio-881 (3c) was active in the neurogenic phase,
while LASSBio-945 (5) and LASSBio-294 (4)18 showed
no activity. This central antinociceptive activity was
confirmed by the hot plate test in mice,28 in which only
LASSBio-881 (3c) was able to increase latency time,
indicating an action at central nervous system level
(Scheme 3). These results suggest distinct mechanisms
of action for compounds LASSBio-881 (3c) and LASS-
Bio-945 (5).
First, we evaluated the in vivo anti-inflammatory pro-
file of 6-nitro-3,4-methylenedioxyphenyl-N-acylhydraz-
one compounds (3a–g) in the carrageenan-induced rat
paw edema,25 aiming to select the more promising com-
pounds. The new NAH compounds (3a–g) and (5),
nimesulide (2) and LASSBio-294 (4), used as reference
compounds, were administered orally at the dose of
300 lmol/kg. The obtained results are disclosed in
Table 2.
LASSBio-881 (3c) and LASSBio-945 (5), which present
3,5-di-tert-butyl-4-hydroxyphenyl group attached to
the imine function of the NAH moiety, were able to
inhibit significantly the edema formation in 31.7% and
Table 2. Anti-inflammatory activity of compounds (3a–g) and (5) in carrageenan-induced rat paw edema assay
Compound
Dose (lmol/kg)
na
DVolumeb (lL)
Inhibition (%)
ID50(lmol/kg)
Vehicle
2 Nimesulide
—
10
5
5
5
5
8
5
8
8
8
8
521.8 25.4
221.4 42.6
366.8 33.3
500.1 42.7
503.5 44.3
356.3 16.0
503.1 39.5
402.8 40.0
452.4 14.4
412.6 22.4
331.8 27.6
—
—
300
300
300
300
300
300
300
300
300
300
57.5*
29.7*
4.1 nsc
3.4 ns
31.7*
3.5 ns
22.8*
13.3 ns
20.9 ns
36.4*
22.7
14.3
—
4 LASSBio-294
3a LASSBio-879
3b LASSBio-880
3c LASSBio-881
3d LASSBio-882
3e LASSBio-934
3f LASSBio-935
3g LASSBio-936
5 LASSBio-945
—
194.7
—
—
—
—
96.5
Compounds were administered orally.
a Number of animals.
b Results expressed in terms of mean SEM.
c Not statistically significant.
* p < 0.05 (ANOVA One Way followed by Dunnet test).

C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
2425
Table 3. Anti-inflammatory profile of compounds (3c) and (5) in murine ear edema assay
Compound
Dose (lmol/kg)
TPA-induced murine ear edema
AA-induced murine ear edema
na
Edema formationb (%)
Inhibition (%)
n
Edema formationc (%)
Inhibition (%)
Vehicle
—
12
6
113.9 14.0
126.0 13.4
125.9 19.4
86.4 8.5
—
13
9
24.5 5.8
ꢀ2.3 2.3
25.1 5.3
15.8 3.0
18.1 3.3
—
100*
2 Nimesulide
4 LASSBio-294
3c LASSBio-881
5 LASSBio-945
300
300
300
300
ꢀ10.6 nsc
ꢀ10.5 ns
24.1 ns
ꢀ19.6 ns
6
10
14
9
ꢀ1.6 ns
36.0 ns
26.7 ns
6
6
136.3 8.4
Compounds were administered orally.
a Number of animals.
b Results expressed in terms of mean SEM.
c Not statistically significant.
* p < 0.05 (ANOVA One Way followed by Dunnet test).
Table 4. Antihyperalgesic profile of compounds (3c–d) and (5) in the formalin test
Compound
Dose (lmol/kg)
na
1st phase (neurogenic)b
time (s)
1st phase
inhibition (%)
2nd phase (inflammatory)b
time (s)
2nd phase
inhibition (%)
Vehicle
—
10
7
66.2 5.0
60.0 2.3
54.6 4.3
36.8 1.7
80.2 6.8
63.5 3.0
—
169.5 6.0
50.3 2.6
87.1 9.8
146.8 4.0
167.0 18.5
164.2 2.6
—
2 Nimesulide
4 LASSBio-294
3c LASSBio-881
3d LASSBio-882
5 LASSBio-945
100
100
100
100
100
9.3 nsc
17.9 ns
44.5*
70.3*
48.6*
13.4 ns
1.5 ns
3.1 ns
10
8
6
ꢀ21.1 ns
4.1 ns
8
Compounds were administered orally.
a Number of animals.
b Results expressed in terms of mean SEM.
c Not statistically significant.
* p < 0.05 (ANOVA One Way followed by Dunnet test).
Scheme 3. Time course effect of compounds (3c) and (5) in the hot plate test. Compounds ere administered orally. Data are expressed as
means SEM (n = 7–10 animals per group). *p < 0.05 (Student’s t test) as compared to the vehicle control group.
These results prompted us to evaluate LASSBio-881 (3c)
on cannabinoid receptors, since its in vivo profile is sim-
ilar to that previously described for AEA (1).6,7 The
in vitro CB1-binding profile was evaluated in murine
brain homogenate toward [3H]SR141716A,29 and results
are depicted in Scheme 4. LASSBio-881 (3c) was able to
displace the radioligand in 71% at 100lM, while LASS-
Bio-945 (4) was not able of doing so (ca. 20%), in com-
plete agreement to their in vivo antinociceptive profile.
With these results in hands, it can be postulated that
LASSBio-881 (3c) presented an antinociceptive action
probably due to its binding toward CB1-receptors,
showing the same profile of AEA (1).
activated spleen T-cells30 in an attempt to verify the
CB2-ligand profile for this compound. Scheme 5 shows
that lymphocyte proliferation31 could be nicely inhibited
by LASSBio-881 (3c), and this response can be mediated
by an action on CB2 receptors, since AEA (1) showed
the same profile. On the other hand, in contrast to
AEA (1), LASSBio-881 (3c) was not able to induce nei-
ther apoptosis nor necrosis30 (Table 5), showing its spec-
ificity in prevention of T-cell proliferation without
provoking cell death. These results enable us to rational-
ize the anti-inflammatory mechanism of LASSBio-881
(3c) as an immunomodulatory response, which can be
promoted by its action on CB2 receptors.
We also evaluated the immunomodulatory profile of
LASSBio-881 (3c) on mitogen (PMA) and anti-CD3
According to the above-mentioned results, the presence
of the nitro group is essential for the central analgesic

2426
C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
only an anti-inflammatory profile and LASSBio-882
(3d) was not anti-edematogenic.
2.3. Electrochemical studies
In order to clarify the relative contributions of the nitro
and 3,5-di-tert-butyl-4-hydroxyphenyl groups to the
redox character, the voltammetric response of LASS-
Bio-881 (3c), as well as of LASSBio-945 (5) and LASS-
Bio-882 (3d), was determined in aprotic media (DMF +
TBAP 0.1 M) using a glassy carbon working electrode,
Pt and AgjAgCl, Clꢀ 0.1 M, as auxiliary and reference
electrode, respectively.32 Table 6 shows both cathodic
and anodic peak potentials for the first reduction (EpcI)
and oxidation (EpaI) steps, respectively, at a sweep rate
of 0.1 V/s.
Scheme 4. Effect of compounds (3c) and (5) in CB1-cannabinoid
binding assay. Data are expressed as means SEM (n = 5 animals per
group). *p < 0.01 (Student’s t test) as compared to the vehicle control
group.
In electrochemical terms, LASSBio-881 (3c) bears mixed
electroactivity, due to the presence of the reducible
nitroaromatic function, together with the oxidizable hin-
dered phenol, both groups interconnected by a NAH
chain. A comparison of LASSBio-881 (3c) with LASS-
Bio-882 (3d), devoid of the phenol function, and with
LASSBio-945 (5), a non-nitrated NAH, could explain
the influence of each group on the behavior of this class
of compounds. The data displayed in Table 6 show that
LASSBio-881 (3c) exhibits a more complex redox pro-
file, displaying peaks in both cathodic and anodic
regions. As seen for LASSBio-945 (5) (Epc1 = ꢀ2.07 V),
NAH sub-unit is also an electroreducible group,33 how-
ever its contribution to reduction potential of both
LASSBio-881 (3c) (Epc1 = ꢀ1.02 V) and LASSBio-882
(3d) (Epc1 = ꢀ1.10 V) is supplanted by the presence of
nitro group, which is more easily reduced. When we
compare the Epc1 values of LASSBio-881 (3c) and
LASSBio-882 (3d), related to the reduction of the
nitroaromatic group, it is evident that the electro-
donating phenol group impairs the nitroreduction
(DEpc1 = 0.08 V), thus reflecting the interaction between
both groups through the NAH moiety. Similar effect is
observed in the anodic region, where the peaks (Epa1)
for LASSBio-881 (3c), LASSBio-945 (5), and LASS-
Bio-882 (3d) appear at +1.04, +0.84, and +1.35 V,
respectively. The anodic waves are related to the oxida-
tion of phenol, in LASSBio-881 (3c) and LASSBio-945
(5), with a DEpc1 = 0.20 V, the first one being oxidized
less easily due to the presence of the electron-withdraw-
ing nitro group. It is not surprising that the electrochem-
ical processes which involve addition or removal of
electrons from an organic framework should correlate
with the ability of substituents to withdraw or supply
electron density to that framework.
Scheme 5. Inhibition of lymphocyte proliferation by AEA (1) and
LASSBio-881 (3c). Each point represents triplicate determinations
and results are based on at least three-independent experiments
(means SEM). *p < 0.01 (Student’s t test) as compared to the vehicle
control group.
Table 5. Induction of T-lymphocyte apoptosis by AEA but not for
LASSBio-881 (3c)
Treatment
% Necrosis % Apoptosis
(Anexine V)
(7-AAD)
Control
Anti-CD3
0.3
0.1
0.1
42
39
Anti-CD3 + AEA (1) 10 lM
Anti-CD3 + LASSBio-881 (3c) 10 lM 0.0
50.8*
43.8
Each point represents triplicate determinations and results are based
on at least three independent experiments (mean SEM).
* p < 0.01 (Student’s t test) as compared to the test group (anti-CD3).
2.4. Antioxidant activity
First, we evaluated the in vitro antioxidant profile of
compound LASSBio-881 (3c) using the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay,34 which measures the
hydrogen-donating ability of antioxidants to convert sta-
ble DPPH free radical to 1,1-diphenyl-2-picrylhydrazine.
The reaction is accompanied by a change in color from
deep-violet to light-yellow and is monitored spectropho-
tometrically. BHT (tert-butylhydroxytoluene) was used
activity, giving to compound LASSBio-881 (3c) the abil-
ity to exert both anti-inflammatory and antinociceptive
activities, as well as the ability to bind to CB1 receptor.
In contrast, it seems that the anti-inflammatory profile is
due to the presence of 3,5-di-tert-butyl-4-hydroxyphenyl
sub-unit, once compound LASSBio-945 (5) presents

C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
2427
as reference antioxidant and results are disclosed in
Table 6. LASSBio-881 (3c) and LASSBio-945 (5) showed
a scavenging activity of 32.0% and 45.7%, respectively, at
100 lM concentration, due to the 3,5-di-tert-butyl-4-
hydroxyphenyl moiety, while LASSBio-882 (3d), pre-
senting a phenyl sub-unit, was not active (7.5% ns). Thus,
the donation of –OH hydrogen of 3,5-di-tert-butyl-4-
hydroxyphenyl sub-unit is imperatively more effective
in terms of antioxidant profile than –NH hydrogen of
NAH, in complete agreement with the results obtained
from cyclic voltammetric studies, that is, the lesser posi-
tive Epa1, the higher antioxidant activity.
Intending to verify the scavenging effect of LASSBio-
881 (3c) over reactive oxygen species, we performed
the hydroxyl-mediated 20-deoxyribose degradation
assay35 and tested its effects on superoxide radical
production by the xanthine/xanthine oxidase system36
(Table 6). Compound LASSBio-881 (3c) was able to
inhibit deoxyribose oxidation by 84.6%, measured as
thiobarbituric acid reactive species (TBARS), therefore
comparable with the antioxidant effect exerted by all
other compounds studied here and the reference com-
pound BHT. Apparently there is no distinguishable
effect of the 3,5-di-tert-butyl-4-hydroxyphenyl sub-unit,
suggesting an important role of the NAH group in the
stabilization of radicals, as previously described.15b On
the other hand, the xanthine/xanthine oxidase-generated
superoxide flux remained unchanged, indicating low
reactivity of LASSBio-881 (3c) and all other compounds
with superoxide radical anion.
2.5. Molecular modeling studies
In order to rationalize the molecular mechanism of
radical stabilization for LASSBio-881 (3c), molecular
modeling studies were performed, using semiempirical
AM1 method37 available in the Spartan 1.0.5 Pro soft-
ware.38 According to the results from the synthesis of
LASSBio-881 (3c) described above, we obtained a
mixture of E and Z-diastereomers, in an approximate
65:35 ratio, measured by NMR.23 In the present study,
we considered both diastereomers and the relative stabil-
ities of corresponding low energy conformers are shown
in Table 7.
It is clear that the syn-E is the most stable conformer of
LASSBio-881 (3c). This result corroborates the experi-
mentally obtained NMR-determined diastereomeric
ratio, showing major formation of the E-diastereomer.
We have noticed the possibility of intramolecular hydro-
gen bonding between the amidic hydrogen and one of
the oxygen atoms from nitro group in both diastereo-
mers in all conformers. Consequently, it was also found
that the NAH chain is not planar in relation to the 1,3-
benzodioxolyl system, that is, dihedral angle between
C6@C5–C10@O of ca. 100ꢁ, as a result of an ortho effect
of nitro group as well as hydrogen bonding between
amidic N–H and the nitro group, with the formation
of a 7-membered ring shape.
With the minimum energy conformers of both E- and Z-
diastereomers in hands, we followed the studies focusing

2428
C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
Table 7. Conformers from systematic conformational analysis for LASSBio-881 (3c)
ꢀ
Conformer AM1 DHf (kcal/mol) Relative stability DDHf (kcal/mol) C6@C5–C10@O dihedral angle (ꢁ) N–Hꢂ ꢂ ꢂ O–N@O distance (A)
˚
syn-E
anti-E
syn-Z
anti-Z
ꢀ67.39
ꢀ62.94
ꢀ65.18
ꢀ66.89
0.00
4.45
2.21
0.50
98.7
102.3
96.7
2.56
2.32
2.37
2.43
98.7
on the stability of radicals of LASSBio-881 (3c). This
compound, as evidenced in the electrochemical studies,
has a dual behavior with the ability of accepting elec-
trons in the nitro group and donating hydrogen atoms
from either amidic N–H or phenolic O–H. Considering
these three possibilities of radical formation, we analyzed
their relative stability, as well as their heats of formation
from the corresponding singlet neutral starting diaste-
reomers. In addition, we have analyzed the single
occupied molecular orbital (SOMO) energies (Table 8).
(DDHf ꢁ ꢀ53 kcal/mol) when comparing to N-(DDHf ꢁ
+32 kcal/mol) and O-radical (DDHf ꢁ +36 kcal/mol).
The energies of SOMO also reveal that the nitro
group, which is able to work as electron acceptor in
single electron transfer (SET) processes,39 has a critical
role in radical formation from neutral LASSBio-881
(3c). Concerning the donation of radical hydrogen
(HÅ) from either amide NAH or phenol hydroxyl
subunits, we can consider these functional groups as
energetically equivalent electron donors, being the
corresponding radical species formed endothermically.
Their corresponding SOMO energy values also show
the same pattern of donating behavior for both
According to these results, the radical anion formation
is energetically favored, as it is an exothermic process
Table 8. Energy profile of radicals generated from neutral LASSBio-881 (3c)
Conformer
AM1 DHf neutral
(kcal/mol)
AM1 DHf
radical anion
(kcal/mol)
ESOMO radical
anion (eV)
AM1 DHf N-radical
(kcal/mol)
ESOMO
N-radical (eV)
AM1 DHf
O-radical
(kcal/mol)
ESOMO
O-radical (eV)
syn-E
anti-Z
ꢀ67.39
ꢀ66.89
ꢀ120.00
ꢀ120.18
ꢀ2.98
ꢀ2.90
ꢀ35.20
ꢀ33.48
ꢀ1.80
ꢀ1.62
ꢀ31.10
ꢀ29.42
ꢀ1.68
ꢀ1.67

C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
2429
groups, which can be an attribute of resonance stabiliz-
ing effect.
of starting material. After 30 min, addition of distilled
water provoked the crystallization of a light yellow
solid, which was filtered and washed with 200 mL of
distilled water and recrystallized from ethanol/water
1
3. Conclusions
(1.24 g, 95%); mp 91–92 ꢁC (lit. 93–94 ꢁC21). H NMR
(200 MHz, DMSO-d6, TMS) d (ppm): 6.34 (s, 2H,
H-2); 7.32 (s, 1H, H-4); 7.74 (s, 1H, H-7); 10.09 (s,
1H, –CHO). RMN 13C (50 MHz, DMSO-d6, TMS) d
(ppm): 104.8 (C-2); 105.4 (C-7); 107.4 (C-4); 127.9 (C-
5); 146.1 (C-6); 151.8 (C-3); 152.3 (C-1); 188.6 (C@O).
IR (emax, KBr) e (cmꢀ1): 1682, 1518, 1368, 1336, 1126,
1119, 1032, 1021.
LASSBio-881 (3c) was identified as a novel central
antinociceptive and peripheral anti-inflammatory com-
pound, acting probably as a non-selective cannabinoid
ligand, in a different manner from its lead compounds
LASSBio-294 (4) and nimesulide (2). In addition, we
observed that LASSBio-881 (3c) was able to bind to
CB1 receptors and also to inhibit T-cell proliferation
probably through the binding of CB2 receptors, in a
non-proapoptotic manner, different from AEA (1).
4.1.2. Methyl 6-nitro-1,3-benzodioxole-5-carboxylate (9).14c
To a solution of (8) (1.5 g, 7.7 mmol) in absolute meth-
anol (10 mL) cooled at 0ꢁC were successively added
methanolic solutions (each 10 mL) of iodine (2.54 g,
23.1 mmol) and KOH (1.3 g, 23.1 mmol) at 0 ꢁC. After
stirring for 1.5 h at 0 ꢁC, small amounts of saturated
NaHSO3 solution were added until the disappearance
of the brown color. Next, the methanol was almost
totally evaporated under reduced pressure. To the resi-
due was added water, and the desired product (9) was
obtained by filtration and recrystallized in ethanol/water
It was also demonstrated that LASSBio-881 (3c) exhib-
ited important antioxidant properties probably due to
its particular redox profile, which reflects its structural
features by the presence of both nitroaryl and 3,5-di-
tert-butyl-4-hydroxyphenyl sub-units.
In addition, we showed that these sub-units are also
essential for the pharmacological activity. We observed
the importance of the nitro group of LASSBio-881
(3c) for the antinociceptive activity, as well as for
the CB1-binding profile, since LASSBio-945 (5) was
inactive. Otherwise, the anti-inflammatory activity of
LASSBio-881 (3c) is probably due to its 3,5-di-tert-
butyl-4-hydroxyphenyl radical scavenging sub-unit,
considering that LASSBio-945 (5) presented only an
anti-inflammatory activity and LASSBio-882 (3d) did
not present anti-edematogenic properties.
1
as a light yellow solid (1.52 g; 88%); mp 102-103 ꢁC. H
NMR (200 MHz, CDCl3, TMS) d (ppm): 3.89 (s, 3H,
OCH3); 6.18 (s, 2H, H-7); 7.03 (s, 1H, H-4); 7.38 (s,
1H, H-2). 13C NMR (50 MHz, CDCl3, TMS) d (ppm):
53.5 (OCH3); 103.8 (C-2); 105.1 (C-7); 108.6 (C-4);
124.0 (C-5); 143.2 (C-6); 149.8 (C-3); 151.5 (C-1);
165.9 (C=O). IR (emax, KBr) e (cmꢀ1): 1717, 1509,
1488, 1362, 1272, 1108, 1038.
4.1.3. 6-Nitro-1,3-benzodioxole-5-carbohydrazide (10).14c
To a solution of 0.5 g (2.22 mmol) of (9) in 2 mL of etha-
nol was added 6.7 mL of 80% hydrazine monohydrate.
The reaction mixture was kept in ambient temperature
for 2 h, when TLC indicated the end of the reaction. Then,
the media were neutralized with concentrated HCl in an
ice bath and the resulting precipitate was filtered out
and recrystallized from ethanol–water, affording the title
compound as a yellow solid (0.35 g, 70%), mp 184–
185 ꢁC. RMN 1H (200 MHz, DMSO-d6, TMS) d (ppm):
4.42 (s, 2H, NH2); 6.26 (s, 2H, H-2); 7.03 (s, 1H, H-4);
7.64 (s, 1H, H-7); 9.59 (s, 1H, NH). RMN 13C (50 MHz,
DMSO-d6, TMS) d (ppm): 104.2 (C-2); 105.3 (C-7);
108.5 (C-4); 128.3 (C-5); 141.9 (C-6); 148.8 (C-3); 151.7
(C-1); 165.2 (C@O). IR (emax, KBr) e (cmꢀ1): 3336,
3199, 3120, 1670, 1519, 1502, 1475, 1328, 1257, 1030,
880.
4. Experimental
4.1. Chemistry
Reactions were routinely monitored by thin-layer chro-
matography (TLC) in silica gel (F245 Merck plates) and
the products visualized with iodine or ultraviolet lamp
1
(254 and 365 nm). H and 13C nuclear magnetic reso-
nance (NMR) spectra were determined in DMSO-d6
solutions using a Bruker AC-200 spectrometer. Peak
positions are given in parts per million (d) from tetra-
methylsilane as internal standard, and coupling constant
values (J) are given in Hertz. Infrared (IR) spectra were
obtained using a Nicolet Magna IR 760 spectrometer.
Samples were examined as potassium bromide (KBr)
disks. Melting points were determined using a Quimis
instrument and are uncorrected. Column chromatogra-
phy purifications were performed using silica gel Merck
4.1.4. General procedure for preparation of 6-nitro-N0-
arylmethylidene-1,3-benzodioxole-5-carbohydrazines (3)
and (5).14c To a solution of 1 mmol of (10) or (10a)
in absolute ethanol (10 mL) containing two drops of
37% hydrochloric acid was added 1 mmol of corre-
sponding aromatic aldehyde derivative. The mixture
was stirred at room temperature for 30 min, then
after extensive precipitation, was visualized. Next, the
mixture was poured into cold water, neutralized with
10% aqueous sodium bicarbonate solution, and the pre-
cipitate formed was filtered out and recrystallized from
ethanol–water.
1
230–400 mesh. All described products showed H and
13C NMR spectra according to the assigned structures.
All organic solutions were dried over anhydrous sodium
sulfate and all organic solvents were removed under
reduced pressure in rotatory evaporator.
4.1.1. 6-Nitro-1,3-benzodioxole-5-carbaldehyde (8).22
A
mixture of 1 g (6.7 mmol) of tritured piperonal (7) and
3.3 mL of concentrated nitric acid was kept at 20–
25 ꢁC, in a water bath, observing complete dissolution

2430
C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
4.1.5. N0-(2-Furylmethylidene)-6-nitro-1,3-benzodioxole-
5-carbohydrazine (3a). The derivative (3a) was obtained
as a yellow solid by condensation of (10) with furfural-
dehyde (0.26 g; 88%), mp 226–228 ꢁC. 1H NMR
(200 MHz, DMSO-d6, TMS) d (ppm): 6.30 (s, 10H, H-
2E/Z); 6.54 (dd, 2H, J = 1.8 Hz, H-400Z); 6.64 (dd, 3H,
J = 1.8 Hz, H-400E); 6.72 (d, 2H, J = 3.4 Hz, H-300Z);
6.95 (d, 3H, J = 3.4 Hz, H-300E); 7.15 (s, 2H, H-40Z);
7.30 (s, 3H, H-40E); 7.74 (d, 7H, J = 4.6 Hz, H-500Z,
H-4E/Z); 7.87 (d, 5H, H-500E, H-7Z); 8.10 (s, 3H, H-
7E); 11.87 (s, 2H, H-20Z); 11.90 (s, 3H, H-20E). 13C
NMR (50 MHz, DMSO-d6, TMS) d (ppm): 104.4 (C-
2); 104.7 (C-7); 105.5 (C-4); 108.5 (C-300); 108.9 (C-300);
112.5 (C-400); 112.7 (C-400); 128.6 (C-5); 134.9 (C-40);
138.1 (C-40); 141.2 (C-6); 145.5 (C-500); 145.9 (C-500);
148.8 (C-200); 149.6 (C-200); 152.2 (C-1a); 152.6 (C-3a);
161.9 (C-10); 167.9 (C-10). IR (emax, KBr) e (cmꢀ1):
3197, 3123, 3061, 2918, 2855, 1644, 1623, 1533, 1504,
1486, 1381, 1293, 1281, 1261, 1164, 1126, 1033, 1017,
939, 877, 763.
hyde (0.30 g; 95%), mp 216–218 ꢁC. 1H NMR
(200 MHz, DMSO-d6, TMS) d (ppm): 6.31 (s, 66H,
H-2E/Z); 7.34 (m, 165H, H-200,300,400,500,600E/Z); 7.45
(s, 10H, H-4Z); 7.72 (s, 23H, H-4E); 7.76 (s, 33H,
H-7E/Z); 7.99 (s, 10H, H-40Z); 8.22 (s, 23H, H-40E);
11.94 (s, 10H, H-20Z); 11.97 (s, 23H, H-20E). 13C
NMR (50 MHz, DMSO-d6, TMS) d (ppm): 104.4
(C-2); 104.6 (C-7); 108.6 (C-4); 108.9 (C-4); 127.1
(C-300,500); 127.7 (C-300,500); 128.4 (C-5); 129.2 (C-
200,600); 129.3 (C-200,600); 130.4 (C-400); 130.7 (C-400);
141.6 (C-6); 144.8 (C-40); 148.8 (C-40); 152.3 (C-1a);
152.7 (C-3a); 162.0 (C-10); 168.0 (C-10). IR (emax
,
KBr) e (cmꢀ1): 3222, 3114, 3059, 2916, 2851, 1646,
1607, 1533, 1503, 1485, 1381, 1279, 1161, 1123, 1033,
928, 878, 768, 693.
4.1.9. 6-Nitro-N0-(2-pyridinylmetilidene)-1,3-benzodioxole-
5-carbohydrazine (3e). The derivative (3e) was obtained
as a yellow solid by condensation of (10) with 2-pyridin-
ecarboxaldehyde (0.26 g; 84%), mp 184–185 ꢁC. 1H
NMR (200 MHz, DMSO-d6, TMS) d (ppm): 6.32 (s,
20H, H-2E/Z); 7.37 (m, 10H, H-500,600E/Z); 7.58 (s,
10H, H-4E/Z); 7.74 (m, 10H, H-7E/Z); 7.91 (m, 10H,
H-400E/Z); 8.03 (s, 3H, H-40Z); 8.24 (s, 7H, H-40E);
8.54 (d, 3H, J = 3.6 Hz, H-300Z); 8.62 (d, 7H,
J = 4.2 Hz, H-300E); 12.15 (s, 3H, H-20Z); 12.19 (s, 7H,
H-20E). 13C NMR (50 MHz, DMSO-d6, TMS) d
(ppm): 104.5 (C-2); 104.6 (C-7); 105.5 (C-4); 108.7
(C-300); 108.9 (C-300); 119.5 (C-500); 120.5 (C-500); 124.8
(C-400); 125.1 (C-400); 127.2 (C-5); 137.3 (C-40); 137.4
(C-40); 144.8 (C-6); 150.0 (C-600); 150.1 (C-600); 153.2
(C-1a); 155.5 (C-3a); 155.9 (C-200); 156.2 (C-200); 165.4
(C-10); 171.3 (C-10). IR (emax, KBr) e (cmꢀ1): 3427,
3198, 3122, 3063, 2983, 2925, 2854, 1663, 1609, 1523,
1505, 1485, 1432, 1368, 1336, 1288, 1262, 1163, 1126,
1032, 921, 874, 777, 762, 640, 583.
4.1.6. 6-Nitro-N0-(2-thienylmethylidene)-1,3-benzodioxole-
5-carbohydrazine (3b). The derivative (3b) was obtained
as a yellow solid by condensation of (10) with 2-thio-
phenecarboxaldehyde (0.29 g; 90%), mp 240–242 ꢁC.
1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 6.30
(s, 10H, H-2E/Z); 7.03 (dd, 2H, J = 3.6 Hz, H-400Z);
7.13 (m, 5H, H-400E, H-300Z); 7.31 (m, 5H, H-300E, H-
40Z); 7.50 (m, 5H, H-40E, H-500Z); 7.71 (m, 8H, H-4E/
Z, H-500E); 8.14 (s, 2H, H-7Z); 8.42 (s, 3H, H-7E);
11.88 (s, 2H, H-20Z); 11.92 (s, 3H, H-20E). 13C NMR
(50 MHz, DMSO-d6, TMS) d (ppm): 104.4 (C-7);
104.5 (C-2); 105.4 (C-4); 108.6 (C-500); 108.9 (C-500);
128.1 (C-5); 128.2 (C-400); 128.3 (C-400); 129.1 (C-300);
129.7 (C-300); 130.9 (C-40); 131.8 (C-40); 141.5 (C-6);
143.4 (C-500); 148.7 (C-1a); 149.1 (C-3a); 152.2 (C-200);
152.6 (C-200); 161.7 (C-10); 167.8 (C-10). IR (emax, KBr)
e (cmꢀ1): 3191, 3073, 3060, 3032, 2992, 2915, 2844,
1639, 1608, 1591,1560, 1531, 1503, 1485, 1380, 1290,
1264, 1222, 1162, 1125, 1033, 1015, 928, 878, 713.
4.1.10. 6-Nitro-N0-(4-pyridinylmetilidene)-1,3-benzodiox-
ole-5-carbohydrazine (3f). The derivative (3f) was
obtained as a yellow solid by condensation of (10)
with 4-pyridinecarboxaldehyde (0.28 g; 89%), mp 202–
203 ꢁC. 1H NMR (200 MHz, DMSO-d6, TMS) d
(ppm): 6.31 (s, 20H, H-2E/Z); 7.32 (d, 6H, J = 4.9 Hz,
H-200,600Z); 7.52 (s, 3H, H-4Z); 7.71 (d, 14H,
J = 5.8 Hz, H-200,600E); 7.77 (s, 7H, H-4E); 7.99 (s, 3H,
H-40Z); 8.23 (s, 7H, H-40E); 8.56 (d, 6H, J = 4.9 Hz,
H-300,500Z); 8.68 (d, 14H, J = 5.8 Hz, H-300,500E); 12.27
(s, 3H, H-20Z); 12.30 (s, 7H, H-20E). 13C NMR
(50 MHz, DMSO-d6, TMS) d (ppm): 104.7 (C-2,7);
108.6 (C-4); 121.2 (C-300,500); 121.7 (C-300,500); 124.9 (C-
5); 141.7 (C-6); 142.2 (C-40); 144.7 (C-400); 145.0 (C-400);
145.8 (C-40); 150.2 (C-200,600); 150.3 (C-200,600); 152.5
(C-1a); 153.4 (C-3a); 167.0 (C-10); 171.5 (C-10). IR
(emax, KBr) e (cmꢀ1): 3450, 3198, 3119, 3050, 2996,
2920, 2857, 1670, 1581, 1530, 1508, 1489, 1421, 1370,
1337, 1298, 1268, 1162, 1125, 1030, 923, 881, 818, 744,
693, 537.
4.1.7. N0-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-6-nitro-
1,3-benzodioxole-5-carbohydrazine (3c). The derivative
(3c) was obtained as a yellow solid by condensation
of (10) with 3,5-di-tert-butyl-4-hydroxybenzaldehyde
(0.36 g; 82%), mp 280–282 ꢁC. 1H NMR (200 MHz,
DMSO-d6, TMS) d (ppm): 1.32 (s, 18H, –CH3Z); 1.40
(s, 36H, –CH3E); 6.29 (m, 6H, H-2E/Z); 7.15 (m, 6H,
H-200,600E/Z); 7.27 (s, 1H, H-4Z); 7.47 (s, 2H, H-4E);
7.67 (m, 1H, H-40Z); 7.73 (s, 2H, H-40E); 7.83 (s, 3H,
H-7E/Z); 11.69 (s, 1H, H-20Z); 11.76 (s, 2H, H-20E).
13C NMR (50 MHz, DMSO-d6, TMS) d (ppm): 30.5
(–CH3); 34.9 (C-Me3); 104.0 (C-7); 104.2 (C-2); 108.7
(C-4); 124.0 (C-5); 124.4 (C-200,600); 128.6 (C-100); 139.5
(C-300,500); 142.0 (C-6); 145.0 (C-40); 148.6 (C-400); 152.5
(C-1a); 156.3 (C-3a); 167.7 (C-10). IR (emax, KBr) e
(cmꢀ1): 3593, 3172, 3065, 2962, 2872, 1662, 1612,
1527, 1509, 1487, 1433, 1401, 1372, 1342, 1259, 1236,
1212, 1139, 1117, 1032, 927, 875, 766.
4.1.11. 6-Nitro-N0-(1H-pyrrol-2-ylmethylidene)-1,3-ben-
zodioxole-5-carbohydrazine (3g). The derivative (3g)
was obtained as a yellow solid by condensation of (10)
with 1H-pyrrol-2-ylcarboxaldehyde (0.27 g; 89%), mp
4.1.8. N0-Benzylidene-6-nitro-1,3-benzodioxole-5-carbo-
hydrazine (3d). The derivative (3d) was obtained as a
yellow solid by condensation of (10) with benzalde-
1
202–203 ꢁC. H NMR (200 MHz, DMSO-d6, TMS) d

C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
2431
(ppm): 6.06 (d, 3H, J = 2.8 Hz, H-400Z); 6.14 (d, 4H,
J = 2.6 Hz, H-400E); 6.29 (s, 17H, H-2E/Z,300Z); 6.47 (s,
4H, H-300E); 6.82 (s, 3H, H-500Z); 6.93 (s, 4H, H-500E);
7.11 (s, 3H, H-40Z); 7.27 (s, 4H, H-40E); 7.70 (s, 3H,
H-4Z); 7.72 (s, 4H, H-4E); 7.86 (s, 3H, H-7Z); 8.07 (s,
4H, H-7E); 10.83 (s, 3H, H-100Z); 11.54 (s, 4H, H-100E);
11.60 (s, 3H, H-20Z); 11.62 (s, 4H, H-20E). 13C NMR
(50 MHz, DMSO-d6, TMS) d (ppm): 104.4 (C-400);
104.8 (C-400); 105.4 (C-2); 108.5 (C-7); 108.9 (C-4);
109.8 (C-300); 109.9 (C-300); 112.1 (C-500); 112.9 (C-500);
117.3 (C-5); 122.5 (C-500); 123.3 (C-500); 131.7 (C-200);
132.2 (C-200); 137.8 (C-40); 141.0 (C-40); 144.9 (C-6);
151.6 (C-1a); 155.3 (C-3a); 164.6 (C-10); 170.6 (C-10).
IR (emax, KBr) e (cmꢀ1): 3478, 3412, 3190, 3113, 3054,
2914, 2855, 1638, 1604, 1536, 1502, 1484, 1427, 1384,
1333, 1298, 1263, 1164, 1127, 1035, 928, 874, 744, 551.
mice were killed by ether inhalation and 7 mm diameter
of right and left ears was cut and weighed. Ear edema
was measured as the differences in weight between the
challenged and the unchallenged ear. Percent inhibition
was calculated by using (C ꢀ T)/C · 100 (%), where C
and T indicate non-treated (vehicle) edema and drug-
treated edema, respectively. Measurements were taken
6h after TPA-induced and 1h after AA-induced ear
edema.
4.2.3. Formalin-induced murine pain model.27 The for-
malin-induced pain test was carried out as described
by Hunskaar and Hole.27 Animals were injected sub-
plantarly with 20 lL of 2.5% formalin in one hind
paw. The compounds were administered orally
(100 lmol/kg in arabic gum 5% as vehicle) 60 min before
formalin injection. The time the mice spent licking or
biting the injected paw or leg was recorded. Two distinct
periods of intensive licking activity were identified and
scored separately unless otherwise stated. The first peri-
od (earlier or neurogenic phase) was recorded 0–5 min
after formalin injection and the second period (later or
inflammatory phase) was recorded 15–30 min after
injection.
4.1.12. N0-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-1,3-
benzodioxole-5-carbohydrazine (5). The derivative (5)
was obtained as a white solid by condensation of (10a)
with 3,5-di-tert-butyl-4-hydroxybenzaldehyde (0.36 g;
1
91%), mp 231–232 ꢁC. H NMR (200 MHz, DMSO-d6,
TMS) d (ppm): 1.33 (s, 18H, –CH3); 5.41 (s, 1H,
–OH); 6.22 (s, 2H, H-2); 6,92 (dd, 1H, J = 8.2 Hz,
0.4 Hz, H-7); 7.15 (s, 2H, H-200,600); 7.37 (dd, 1H,
J = 1.7 Hz, 0.4 Hz, H-4); 7.51 (dd, 1H, J = 8.2 Hz,
1.7 Hz, H-6); 8.06 (s, 1H, H-40); 11.87 (s, 1H, H-20).
13C NMR (50 MHz, DMSO-d6, TMS) d (ppm): 30.7
(–CH3); 35.0 (C-Me3); 102.3 (C-2); 108.0 (C-4); 108.6
(C-7); 123.2 (C-6); 124.4 (C-200,600); 126.2 (C-100); 127.9
(C-5); 139.7 (C-300,500); 147.9 (C-40); 149.5 (C-400); 150.5
(C-3a); 156.6 (C-1a); 162.5 (C-10).
4.2.4. Hot plate test in mice.28 Central analgesic activity
was investigated using the hot plate test as previously
described.28 Swiss mice of both sexes (18–25 g) were
used, maintained with water ad libitum, and fasted for
8 h. In these experiments, the hot plate apparatus
(Ugo Basile, Model-DS 37) was maintained at
55 1 ꢁC. Mice were placed on the heated surface at
0, 30, 60, 90, and 120 min after oral administration of
vehicle, morphine (39.5 lmol/kg, used as positive con-
trol) or test compounds (100 lmol/kg) and the time
between placement and the first sign of paw licking or
jumping was recorded as latency. The basal latencies
were found to be 6–10 s. A cut-off time of 25 s was fol-
lowed to prevent any injury to the paws.
4.2. Pharmacology
4.2.1. Carragenaan-induced rat paw edema assay.25
Fasted Wistar rats of both sexes (150–200 g) were used.
Compounds were administered orally (300 lmol/kg;
0.1 mL/20 g) as a suspension in 5% arabic gum in saline
(vehicle). Control animals received an equal volume of
vehicle. One hour later, the animals were injected with
either 0.1 mL of 1% carrageenan solution in saline
(0.1 mg/paw) or sterile saline (NaCl 0.9%), into the sub-
plantar surface of one of the hind paws, respectively.
The paw volumes were measured 3 h after the subplan-
tar injection using a glass plethysmometer coupled to a
peristaltic pump. The edema was calculated as the
volume variation between the carrageenan- and saline-
treated paw. Nimesulide (300 lmol/kg) was used as stan-
dard drug in the same conditions. Anti-inflammatory
activity was expressed as % of inhibition of the edema
when compared with the vehicle control group.
4.2.5. CB1-binding profile in murine brain homogenate
toward [3H]SR141716A.29 Swiss mice weighing between
30 and 40 g were used. Animals were sacrificed by
decapitation, taking off the whole brain for the assay.
Brains were homogenized in buffer A (320 mM sucrose,
50 mM Tris–HCl, 1 mM EDTA, and 5 mM MgCl2, pH
7.4) in a glass potter (10 movements). For 4 brains,
20 mL of buffer A (5 mL to homogenize and 15 mL to
rinse) was used. The homogenate was centrifuged at
5400 rpm for 10 min at 4 ꢁC. The supernatant was
saved, and the pellet washed once in 10 mL of buffer
A with subsequent centrifugation. The combined super-
natants were centrifuged at 21,000 rpm for 1h at 4 ꢁC.
The pellet was resuspended in 1 mL of buffer B
(50 mM Tris–HCl, 1 mM EDTA, and 3 mM MgCl2,
pH 7.4) to yield a protein concentration of approximate-
ly 4–6 mg/mL. Binding was initiated by the addition of
160 lg membrane protein to silanized tubes containing
50 lL (10 nM) PMSF (amidase-FAAH inhibitor),
50 lL (10 nM) of [3H]SR141716A, 50 lL of vehicle
4.2.2. AA- and TPA-induced murine ear edema.26 Ear
edema was induced by administration of arachidonic
acid (AA—500 lg/ear) or 12-O-tetra-decanoylphorbol
13-acetate (TPA—2 lg/ear) in fasted Swiss mice weigh-
ing 28–25 g. AA and TPA were dissolved in acetone
and 20 ll of solution was applied to the inner and outer
surface of the right ear of the mice. The left ear received
acetone, delivered in the same manner. Treatments were
given orally at a dose of 300 lmol/kg (suspension in ara-
bic gum 5%) 1 h before TPA or AA application. Finally,
(0,1
%
DMSO + buffer C) or 50 lL AM281,
SR141716A analog (CB1 receptor antagonist, 100 lM)
or testing compound and a sufficient volume of buffer

2432
C. D. Duarte et al. / Bioorg. Med. Chem. 15 (2007) 2421–2433
C (50 mM Tris–HCl, 1 mM EDTA, 3 mM MgCl2, and
2.5 mg/mL fatty acid-free BSA, pH 7.4) to bring the
total volume to 500 lL. The addition of 100 lM unla-
beled AM281 was used to assess non-specific binding.
Following incubation (30 ꢁC for 1 h), binding was termi-
nated by the addition of 1 mL of ice-cold buffer D
(50 mM Tris–HCl, pH 7.4, plus 1 mg/mL BSA) to com-
plete volume to 1.5 mL, and vacuum filtration. Filters
should be pre-treated with polyethyleneimine (0.1%)
for at least 2 h. Tubes were rinsed with 2 mL of ice-cold
buffer D, which was also filtered, and the filters were
subsequently rinsed twice with 4 mL of ice-cold buffer
D. Filters were shaken for 1 h in 4 mL of scintillation
fluid, before radioactivity was quantified by liquid
scintillation spectrometry.
the reaction mixture was vigorously shaken at ambient
temperature. DPPH absorption was spectrophotometri-
cally measured at 514 nm. Mean values were obtained
from triplicates.
4.3.3. Hydroxyl-mediated deoxyribose degradation.35
Reaction mixtures (0.5 mL) containing 2-deoxy-D-ribose
(10 mM), Hepes, pH 7.2 (10 mM), test compounds
(10 lL) dissolved in DMSO (0.5 mM), and H2O2
(1 mM) were incubated at 37 ꢁC for 1 h. The reaction
was started by addition of Fe(NH4)2(SO4)2.6H2O
(10 lM). The controls were run in the absence of 2-de-
oxy-D-ribose. Thiobarbituric acid (1% in 50 mM NaOH,
0.5 mL) and o-phosphoric acid (4%, 0.5 mL) were then
added and the reaction mixture was heated at 90–
100 ꢁC for 15 min and cooled, and the absorbance was
determined at 532 nm (19–21). The molar extinction
coefficient used to determine the thiobarbituric acid
reactive substance (TBARS) concentration was
4.2.6. Cell proliferation assay.30 Enriched T cells were
obtained by nylon wool filtration of splenocytes29 deplet-
ed of RDC by treatment with Tris-buffered ammonium
chloride. Nylon wool-enriched T cells were resuspended
in DMEM (Invitrogen, Life Technologies), supplement-
ed with 2 mM glutamine, 5 · 10ꢀ5 M 2-Me, 10 lg/mL
gentamicin, 1 mM sodium pyruvate, and 0.1 mM
MEM non-essential amino acids plus 1% of nutridoma
(Roche). Cultures (0.2 mL) were established in triplicate
in 96-well flat-bottomed microtiter plates (TPP-Germa-
ny) with or without soluble anti-CD3 (2% v/v of
145.2C11 mAb), and PMA (0.5 ng/mL, Sigma) in the
presence of the indicated compound. Cultures were
incubated for 3 days at 37 ꢁC in a humidified atmosphere
containing 7% CO2. The cells were pulsed with [3H]thy-
midine (Amersham Co., UK; 1.0 lCi/well) over the
last 18 h and harvested onto glass fiber filter mats.
Radioactive incorporation into DNA was determined
by liquid scintillation spectrometry.
1,56 · 105 Mꢀ1 cmꢀ1
.
4.3.4. Superoxide scavenging.36 Compounds (0.25 mM)
dissolved in DMSO were added to phosphate buffer
(50 mM, pH 7.8, 37 ꢁC) containing xanthine (0.25 mM),
EDTA (0.10 mM), and cytochrome c (12 lM). Reaction
was started by adding xanthine oxidase (ꢁ0.2 U/mL, in
EDTA 0.1 mM). Reduction rates of cytochrome c by
superoxide anion were measured spectrophotometrically
at the Soret band and expressed as DA/min.
4.4. Molecular modeling
Systematic conformational analysis of NAH com-
pound LASSBio-881 (3c) diastereomers was performed
using semi-empirical Hamiltonian AM1 method37
within SPARTAN 1.0.5 Pro software38 running on
Pentium IV 1.5 GHz. Lowest energy conformers for
each diastereomer were used to evaluate the radical
energy profile.
4.2.7. T cell death assay.31 Nylon wool-enriched T cells
were adjusted to 106 cells mlꢀ1 in DMEM plus 1%
nutridoma, distributed in 24-well plate (1 mL, TPP
Switzerland) and cultured with medium only or were
stimulated with anti-CD3 (145.2C11 mAb) in the pres-
ence of PMA, and with or without LASSBio-881 (3c)
or anandamide (AEA). After 48 h at 37 ꢁC and 7%
CO2 in a humid atmosphere, cells were collected and
stained with FITC-annexin V (PharMingem, BD) and
7-AAD (Calbiochem, CA) and then analyzed by flow
cytometry (FACScalibur, BD).
Acknowledgments
The authors thank CAPES (BR), CNPq (BR), FAPEAL
(BR), FAPERJ (BR), FAPESP (BR), PRONEX (BR),
and IM-INOFAR (BR, #420015/05-1) for financial sup-
port and fellowships (to C.D.D., J.L.M.T., D.I.L.,
M.V.M., F.D., E.J.H.B., F.S.P., M.O.F.G., J.B.C.,
A.L.P.M., C.A.M.F., E.J.B.).
4.3. Antioxidant assays
4.3.1. Cyclic voltammetry.32 Electrochemical studies
were carried out in dimethylformamide (DMF) contain-
ing 0.1 M tetrabutylammonium perchlorate (TBAP),
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