Design and synthesis of 3,4-methylenedioxy-6-nitrophenoxyacetylhydrazone derivatives obtained from natural safrole: New lead-agents with analgesic and antipyretic properties

Article abstract of DOI:10.1016/j.bmc.2006.07.046

In this work, we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and antipyretic properties of new 2-(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhydrazone derivatives (3), designed exploring molecular hybridization and isosteric replacement approaches between nimesulide (1) and carbanalogue NAH series (2) developed at LASSBio. Target compounds were synthesized in very good yields exploiting abundant Brazilian natural product safrole (4) as starting material. The evaluation of the antinociceptive properties of this series led us to discover a new potent prototype of analgesic and antipyretic agent, that is, NAH derivative 3c, named LASSBio-891, which showed to be more potent than dipyrone used as standard.

Full text of DOI:10.1016/j.bmc.2006.07.046

Bioorganic & Medicinal Chemistry 14 (2006) 7924–7935
Design and synthesis of 3,4-methylenedioxy-6-
nitrophenoxyacetylhydrazone derivatives obtained from natural
safrole: New lead-agents with analgesic and antipyretic properties
Heleno J. C. Bezerra-Netto,a,b Daniel I. Lacerda,a,c Ana Luisa P. Miranda,a,c
a,d
Helio M. Alves, Eliezer J. Barreiroa,b and Carlos A. M. Fragaa,b,
*
´
a
ˆ
Laborato´rio de Avaliac¸a˜o e Sı´ntese de Substancias Bioativas (LASSBio), Faculdade de Farmacia,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, PO Box 68023, RJ 21944-971, Brazil
´
b
ˆ
´
Programa de Mestrado em Ciencias Farmaceuticas, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro,
ˆ
Rio de Janeiro, RJ, Brazil
ˆ
c
Programa de Po´s-Graduac¸a˜o em Farmacologia e Terapeutica Experimental, Departamento de Farmacologia Basica e Clınica,
´ ´
ˆ
´
Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
dDepartamento de Produtos Naturais e Alimentos, Faculdade de Farma´cia, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, Brazil
Received 5 June 2006; revised 14 July 2006; accepted 26 July 2006
Available online 10 August 2006
Abstract—In this work, we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and antipyretic properties of
new 2-(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhydrazone derivatives (3), designed exploring molecular hybridization and isosteric
replacement approaches between nimesulide (1) and carbanalogue NAH series (2) developed at LASSBio. Target compounds were
synthesized in very good yields exploiting abundant Brazilian natural product safrole (4) as starting material. The evaluation of the
antinociceptive properties of this series led us to discover a new potent prototype of analgesic and antipyretic agent, that is, NAH
derivative 3c, named LASSBio-891, which showed to be more potent than dipyrone used as standard.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
One of the most important pain-inducer factors is the
inflammation process that frequently occurs in response
to a tissue damage, resulting in a series of characteristic
aspects correlated with the evolution of the disease, that
is, fever, lethargy, anorexy, and generalized pain in mus-
cles and joints as well as an arisen of the hypersensibility
to pain near the damaged place.3
Pain is an unpleasant and subjective sensation resulting
from a harmful sensorial stimulation that alerts the
body about a current or potential damage to its tissues
and organs.1 It is estimated that more than 75 million
people refer to health services annually, presenting some
form of recurrent or persistent pain.2 In spite of painful
sensation can be solved most efficiently by removal of
the underlying cause, the pain-causing stimulus cannot
always be either easily defined or quickly removed.
Therefore, the health professionals are usually faced
with the necessity to manage the symptomatology of
the pain.1
Among other mediators, the prostaglandins produced in
the initial period of the inflammatory injury are respon-
sible for the local vasodilatation and potentialization of
the edema, leading to the development of redness and
heat in the inflamed tissue.3 In special, prostaglandins
E2 (PGE2) and I2 (PGI2) contribute to the development
of the hyperalgesia associated with the inflammatory
process.4
The ability of non-steroidal anti-inflammatory drugs
(NSAIDs) to modulate the pain, inflammation, and
fever made them one of the most used therapeutical
classes in the world.5 The discovery in 1992 of a second
Keywords: N-Acylhydrazone; Safrole; Analgesic activity; Antipyretic
activity; 1,3-Benzodioxole derivatives; Privileged structure.
*
Corresponding author. E-mail: cmfraga@pharma.ufrj.br
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.046

H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
7925
isoform of the cyclooxygenase (COX-2), related to the
bioformation of prostaglandins in inflamed sites, con-
tributed to the sprouting of a new class of NSAIDs,
which act as selective COX-2 inhibitors, with lower side
effects.6
most active compound of this family, that is, para-dim-
ethylamino derivative (2a), was able to inhibit in 67%
the acetic acid-induced abdominal constrictions in mice,
having presented a superior inhibitory profile when
compared with that displayed by the classical standards
dipyrone and indomethacin at the same dose, that is,
36% and 56%, respectively.12 A proposal for the molec-
ular mechanism of COX inhibition by hydrazone deriv-
atives was described by Mahy et al.,13 which have
identified an isosteric relationship between benzo-bis-
azaallyl moiety of the arylhydrazones and the bis-allyl
methylene subunit of the arachidonic acid. This struc-
tural similarity seems to be the primary reason for
molecular recognition of the corresponding frameworks
by the target enzyme, in both structures.
Among the most potent drugs that appeared in the ini-
tial phase of the development of selective COX-2 inhib-
itors are the sulides, such as nimesulide (1), which was
launched in 1985, before the COX-2 discovery, as an
important non-steroidal anti-inflammatory (NSAI)
agent without gastric effects.7 At that moment, the ther-
apeutical profile of (1) was attributed to its radical scav-
enger behavior, which may be explained by the presence
of the para-nitrosulfonylaminophenyl unit in its struc-
ture, pharmacophoric for its redox properties.8 Later,
this compound was analyzed in relation to its action
on COX isoforms, having been shown to be 1400-fold
more selective for COX-2.9
In the course of an ongoing research program aimed at
the design of new bioactive compounds acting at the
arachidonic acid cascade enzyme’s level, we described
in this manuscript the design, the synthesis, and the
pharmacological evaluation of a new series of function-
alized 2-(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhyd-
razone (NBNAH) derivatives (3a–j),14 exploring
safrole (4), an abundant Brazilian natural product,15
as starting material (Fig. 1).
Considering this panorama, our laboratory had synthe-
sized structurally diverse N-acylarylhydrazone (NAH)
derivatives presenting important analgesic, anti-inflam-
matory, and anti-thrombotic profiles.10,11 In this con-
text, the analgesic activity of 1,3-benzodioxolyl NAH
series (2) was characterized.12 indicating to us the bio-
phoric characteristics of these new prototypes of power-
ful antinociceptive drug candidates. For instance, the
The title compounds (3) were structurally designed
through the isosteric exchange16 of the benzylic
Figure 1. Design concept of new 2-(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhydrazone (NBNAH) derivatives (3a–j).

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H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
methylene group (C, Fig. 1) of N-acylhydrazone series (2)
by an oxygen atom, in order to evaluate any eventual
influence of the conformational restriction in the pharma-
cophoric NAH side chain, promoted by a possible intra-
molecular H-bond formation, in the antinociceptive
profile. Moreover, the nitro group was introduced at
C-6 position of the 1,3-benzodioxole ring in order to
investigate its internal ‘catalytic’ effect in the formation
of free radicals on the N-acylhydrazone moiety, intending
to improve the radical scavenger profile in these new com-
pounds,8 aiming the optimization of its pharmacological
profile through the selective modulation of its redox prop-
erties,13,17 The substituents of the terminal phenyl moiety,
attached at the imine function of NAH derivatives (3),
were rationally elected in order to acquire information
about the influence of electronic and physical–chemical
parameters on the pharmacological activity.
derivative 3d exploiting Oxoneꢁ (2KHSO5ÆKHSO4ÆK2-
SO4) supported in alumina27 or 3-chloroperoxybenzoic
acid (MCPBA) in dichloromethane28 (Scheme 2).
The next step of this work was to determine the relative
configuration of the imine double bond of N-acyl-
arylhydrazone derivatives (3a–j), in order to assure the
diastereomeric ratio essential to the complete under-
standing of the biological effects. The careful analysis
1
of the H NMR spectra of (3a–j) allowed us to detect
the presence of two singlet signals related to the imine
hydrogen, which were attributed to the respective (E)
and (Z)-diastereomers. The presence of both diastereo-
mers in this series of NAH derivatives (3) is in agree-
ment with previous results from our laboratory that
indicated a similar behavior in other NAH series pre-
senting a C2 spacer unit between 1,3-benzodioxole ring
and the acyl moiety.10,12,29,30 Studies using NOESY
1
Additionally, in order to study and validate the pharma-
cophoric character of the N-acylhydrazone and nitro
moieties related to anti-inflammatory and analgesic
activities of the new compounds (NBNAH, 3), we also
elect as target the 5-(benzyloxy)-6-nitrobenzo[1,3]diox-
ole derivative (NBB, 5), 2-(benzo[1,3]dioxol-5-yloxy)-
acetylhydrazone derivative (BNAH, 7), and 5-(benzyl-
oxy)-benzo[1,3]dioxole derivative (BB, 6), designed by
molecular simplification of series (3) by removing,
respectively, the N-acylhydrazone moiety (A), the nitro
group, and both functionalities (Fig. 1).
and COSY techniques of bidimensional H NMR31
did not achieve success in the identification of the rela-
tive configuration of the diastereomers, probably due
to the conformational flexibility around amide bond.
The assignment of the relative configuration of the dia-
stereomers of NAH derivatives (3a–j) was made in
agreement with previous results obtained by Karabatsos
et al.,32–34 which describes that imine-attached hydrogen
signal of (E)-diastereomer is downfielded by 0.2–
0.3 ppm from the corresponding hydrogen atom signal
in (Z)-diastereomer. Therefore, after careful analysis of
1
the H NMR spectra of the mixture of diastereomers
of NAH derivatives (3a–j), we were able to evidence that
the major one presents (E) configuration similar to that
we have found for the N-acylhydrazones of the previous
series (2).12 In order to obtain additional evidences
about the relative configuration of the imine double
bond of nitro-acylarylhydrazone derivatives (3a–j), we
performed a brief molecular modeling study employing
the semi-empirical AM1 method35 in the Spartan Pro
1.5.0 program36 using the non-substituted derivative
(3c) as model (Fig. 2). Systematic conformational anal-
ysis was carried out to determine the heat formation
value of the more stable diastereomeric form. For deriv-
ative (3c), the obtained value for the more stable con-
former of the (E)-diastereomer was ꢀ33.75 kcal/mol,
while the energy found for the corresponding (Z)-diaste-
reomer was ꢀ31.27 kcal/mol (Fig. 2). Although the cal-
culated difference in heat formation seems to be
insufficient to let us to identify unambiguously, what
diastereomeric form of (3c) predominates, these studies
indicated that (E)-diastereomer, presenting minor heat
formation, could be preferentially formed.
2. Chemistry
The obvious synthetic route planned to obtain the desired
NBNAH derivatives (3a–j) is shown in Scheme 1. The new
NAH derivatives of series (3) were prepared from pipero-
nal (8), obtained in ca. 75% overall yield from the natural
safrole (4),18 using base-catalyzed isomerization of the
terminal double bond followed by oxidative cleavage.19
Performing a Bayer–Villiger oxidation of the aromatic
aldehyde (8) through its treatment with performic acid
generated ‘in situ,’ we were able to obtain the correspond-
ing phenolic derivative sesamol (9) in 90% yield.20 Next,
the aryl-ether derivative (10) was prepared, in 85% yield,
exploring the O-alkylation of (9), by treating the pheno-
late intermediate formed in basic conditions with ethyl
2-bromoacetate.21 Next, regioselective nitration at C-6
of 1,3-benzodioxole ring of the derivative (10) with nitric
acid in chloroform22 furnished the nitro-ester derivative
(11) in high yield, which after treatment with hydrazine
hydrate in ethanol12,23–26 resulted in the formation of
the corresponding hydrazide (12), the key-intermediate
of this synthesis route, in 85% yield.
The ratio between (E)/(Z) diastereomers could be de-
fined from the relative integration of imine-attached
1
hydrogen in the corresponding H NMR spectra (Table
Finally, the new safrole-derived NBNAH derivatives
(3a–h) were obtained, in good yields, by condensing
hydrazide derivative (12) with different aromatic alde-
hydes (ArCHO) in ethanol, using hydrochloric acid as
catalyst.12,23,25,26
1) and was confirmed by reversed-phase high perfor-
mance liquid chromatography (RP-HPLC), also
employing the acylhydrazone derivative (3c) as model
compound (Fig. 3). The RP-HPLC analysis has demon-
strated a ratio between (E)/(Z)-diastereomers of 2.8:1, in
agreement with the relationship that we have obtained
by 1H NMR (see Table 1). In order to explain the polar-
ity difference between the (E) and (Z)-diastereomers of
The methylsulfone (3i) and methylsulfoxide (3j) NAH
derivatives were, respectively, prepared by chemoselec-
tive oxidation of the corresponding thiomethyl NAH

H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
7927
Scheme 1. Reagents and conditions: (a) KOH aq 3 N, n-BuOH, room temperature, 3 h, 98%; (b) i—O3/O2, AcOH, 0 ꢂC, 1 h; ii—Zn0, AcOH, 93%
(75%, three steps); (c) i—30% H2O2, HCO2H, CH2Cl2, reflux, 18 h; ii—NaOH 1,5 N, room temperature, 15 min, 77%; (d) K2CO3, DMF, then
2-bromoethyl acetate, room temperature, 20 h, 85%; (e) HNO3, CHCl3, 0 ꢂC, 3 h, 90% (11) and 75% (5); (f) NH2NH2ÆH2O 80%, EtOH, room
temperature, 5 min, 85%; (g) Ar-CHO, EtOH, 37% aq HCl (cat.), room temperature, 30 min; (h) K2CO3, DMF, then benzyl bromide, 20 h, 95%.
3c in the HPLC (Fig. 3), the dipole moment (l) of the
minimum energy conformers of each diastereomer was
calculated using the Hartree–Fock ab initio method, in
the program Spartan 1.5.0 Pro.36 The correlation be-
tween the obtained l values, that is, 6.91 for (E)-diaste-
reomer and 7.22 for (Z)-diastereomer, with the elution
order evidenced by HPLC, corroborated that the major
diastereomer of the mixture should present the relative
(E)-configuration.
regioselectively nitrated with nitric acid (67%) in chloro-
form at 0 ꢂC, resulting in the formation of the corre-
sponding nitroarylether derivative (5) in 75% yield.22
Thereafter, treatment of previously obtained aryl-ether
derivative (10) with hydrazine hydrate gave the corre-
sponding hydrazide intermediate, which after acid-cata-
lyzed condensation with benzaldehyde furnished the
desired BNAH derivative (7), in 59% overall yield (two
steps)12,23–26 (Scheme 1).
Next, the target compounds 5, 6, and 7 were prepared
from natural safrole (4), by applying the synthetic route
illustrated in Scheme 1. Alkylation of sesamol (9) with
benzyl bromide furnished the expected arylbenzyl-
ether (6) in 95% yield, using the same experimental pro-
tocol described previously.21 Next, derivative (6) was
3. Pharmacological results
The analgesic and anti-inflammatory profiles of new
NBNAH derivatives (3a–j) and its structurally simplified

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H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
Scheme 2. Reagents and conditions: (a) Oxoneꢁ, alumina, MeOH, reflux, 8 h, 85%; (b) MCPBA, CH2Cl2, 0 ꢂC, 3 h, 80%.
Figure 2. Minimum energy conformers of the diastereomers of NAH derivative (3c) obtained by applying semiempirical AM1 method.
Table 1. Physical and spectral properties of the NBNAH derivatives (3a–j)
Compound
Molecular formulaa
MW
Yield (%)
Mp (ꢂC)
d (ppm)c N@CH
Ratio (E)/(Z)d
(E)
(Z)
3a
3b
3c
3d
3e
3f
C
18H18N4O6
386.36
422.19
343.29
389.07
357.32
373.32
471.50
421.38
405.38
98
95
98
92
62
98
98
85b
80b
222–224
238–240
220–222
210–212
198–200
238–240
248–250
184–186
230–232
8.09
8.23
8.25
8.19
—
7.86
7.97
8.00
7.95
1.8/1
1.2/1
2.8 /1
2.1/1
1.8/1e
2.3/1
1.7/1
2.1/1e
2.1/1e
C16H12BrN3O6
C16H13N3O6
C
17H15N3O6S
C17H15N3O6
C17H15N3O7
C24H29N3O7
8.18
8.16
7.94
7.90
3g
3i
C17H15N3O8S
C17H15N3O7S
7.95–7.61 (m)
8.31–7.70 (m)
3j
a The analytical results for C, H, N, and S were within 0.4% of calculated values.
b Obtained by chemoselective oxidation of the corresponding methylsulfide derivative (3d) as described in Scheme 2.
c Data obtained at 200 MHz, using DMSO-d6 as solvent.
d Data obtained by relative integration of the corresponding imine-attached hydrogen of the (E)- and (Z)-diastereomers.
e Data obtained by relative integration of methyl group-attached hydrogens of the corresponding (E)- and (Z)-diastereomers.
analogues NBB (5), BB (6), and BNAH (7) (Fig. 1) were
evaluated using, respectively, the classical tests of acetic
acid-induced abdominal constrictions in mice37 and car-
rageenan-induced rat paw edema (CIRPE),38 employing
dipyrone and nimesulide as standards. The obtained
results are illustrated in Figures 4 and 5, respectively.
non-substituted analogue (3c), that were able to reduce
remarkably the AcOH-induced constrictions, presenting
59.8% and 57.8% of inhibition, respectively. This profile
is in agreement with that observed for the non-nitrated
carbanalog (2a) (67.1% of inhibition) described previ-
ously,12 indicating, in comparison with compound
(3a), that no significative difference in the antinocicep-
tive activity could be evidenced. However, a great differ-
ence in the analgesic profiles of compound (3b) and
its non-nitrated carbanalog (2b) (non-significative
The analysis of these results allowed us to evidence that
the most active analgesic compounds were the 4-dimeth-
ylamino derivative (3a) and its corresponding

H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
7929
Figure 3. HPLC chromatogram of (E)/(Z)-diastereomeric mixture of
NBNAH derivative 3c. The retention time found for (Z)-diastereomer
was 22.5 min, while the major (E)-diastereomer elutes after 25.0 min.
Figure 5. Anti-inflammatory effects of NBNAH derivative (3c), NBB
derivative (5), BB derivative (6), BNAH derivative (7), and nimesulide
(Nim). All compounds were administered i.p. at a dose of 300 lmol/kg.
Results are expressed as means SEM for n = 10 animals. % of
inhibition was obtained by comparison with vehicle control group
(data not shown). *p < 0.05 (Student’s t-test).
inhibition), as well as between compound (3c) and its
non-nitrated carbanalog (2c) (25.9% of inhibition), was
observed (Fig. 4). These characteristics allow us to state
that the introduction of the nitro moiety and the isoster-
ic replacement in the spacer subunit of NAH derivatives
of series (2) generating the new NAH series (3) produced
strong beneficial effect over its analgesic profile. In spite
of that, we did not have any structural elements to infer
which modification was responsible for the general in-
crease in the antinociceptive activity of the NBNAH
derivatives (3). In order to clarify this point, the analge-
sic profile of compound (7), non-nitrated analogue of
(3c), was also assayed in the same pharmacological pro-
tocol presenting only 12.9% of inhibition at the same
dose (Fig. 4). This expressive reduction of the analgesic
activity has indicated to us the pharmacophoric
character of the ortho-nitrophenyl framework for the
antinociceptive profile of the new NBNAH derivatives
of series (3).
In addition, upon removal of both nitro and acylhydraz-
one moieties from the structure of (3), the analgesic
activity of compound (6) (13.2%) was abolished, con-
firming the importance of these two subunits for the
antinociceptive profile of the NBNAH derivatives (3).
Furthermore, the unsubstituted NAH derivative (3c)
was able to inhibit dose-dependently (5, 10, 50, 100,
and 300 lmol/kg) the acetic acid-induced abdominal
constrictions with an ID50 value of 14.48 lmol/kg which
is ten times more potent than the standard analgesic
drug dipyrone (ID50 = 144.5 lmol/kg), in the same phar-
macological protocol.
The anti-inflammatory profile of NBNAH derivatives
(3a–j), NBB (5), BB (6), and BNAH derivative (7) was
evaluated in the CIRPE test at a dose of 300 lmol/
kg38 (Fig. 5).
Additionally, the suppression of the N-acylhydrazone
subunit in the structure of the derivatives does not elim-
inate the analgesic activity in total but promotes its
significative reduction as evidenced by the direct com-
parison of the antinociceptive profile of BB derivative
(5) (35.6%) with that of the NAH-containing structural
analogue (3c) (57.8%).
The NAH derivatives of series (3) presented a poor anti-
inflammatory profile (data not shown), excepting for the
unsubstituted derivative (3c) (42.8%) identified as the
most active one, suggesting that the elected para-substit-
uents introduced at terminal phenyl ring do not play in
Figure 4. Analgesic effects of NBNAH derivatives (3a–j), NBB (5), BB (6), BNAH (7), dipyrone, and nimesulide on the acetic acid-induced (0.6%, ip)
abdominal constrictions in mice. All compounds were administered p.o. at a dose of 100 lmol/kg. Results are expressed as means SEM for n = 10
animals. % of inhibition was obtained by comparison with vehicle control group (data not shown). *p < 0.05 (Student’s t-test).

7930
H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
favor of the anti-inflammatory activity observed
through this pharmacological protocol.
Moreover, considering the well-known antipyretic effect
of analgesic drugs like dipyrone, we decided also to
investigate the behavior of the two more potent nitro-
acylhydrazone derivatives (3a and 3c) in lipopolysaccha-
ride-induced fever model,40 as depicted in Figure 7. The
results permitted us to evidence the potent and long-last-
ing antipyretic effect of the non-substituted acylhydraz-
one derivative (3c), which displayed a similar profile to
the standard drug dipyrone in reversing the LPS-in-
duced fever at the fourth and fifth hour of the assay.
However, in spite of dipyrone not being more able to re-
duce the rectal temperature of the animals treated with
LPS at the sixth hour of the experiment, compound 3c
remains to present an expressive antipyretic effect p.o.
at the same molar concentration.
On the other hand, the comparison between the anti-
edematogenic activity of compound (3c) with that of
the NAH-suppressed analogue (5) (2.26% of inhibition)
could demonstrate unambiguously the pharmacophoric
character of the acylhydrazone moiety for the anti-
inflammatory profile, as previously described for other
NAH series developed in our laboratory.11
Additionally, in order to investigate the relevance of the
nitrophenyl group for the anti-inflammatory profile of
nitro-acylhydrazone derivative (3c), BNAH compound
(7) was comparatively assayed in the CIRPE test. This
compound was able to inhibit by 54.0% the edema indi-
cating that, in spite of ortho-nitrophenyl moiety has a
pharmacophoric character for the analgesic profile of
the NBNAH derivatives (3), its presence does not seem
to interfere with the anti-inflammatory profile of these
new NAH derivatives.
Among the new NAH derivatives (3), designed by
hybridization strategy and isosteric replacement of the
initial elected prototype (2), was discovered benzyli-
dene-2-(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhydr-
azine (3c), named LASSBio-891, which showed to be ten
times more potent than dipyrone as analgesic agent.
This compound, synthesized in 43% overall yield from
Brazilian abundant natural product safrole (4) by apply-
ing classical methodology, represents a new lead-com-
pound for an antinociceptive and antipyretic drug.
Comparing the anti-edematogenic profile obtained with
the simplified analogue (6), we can infer, once again, the
great importance of the NAH framework for the anti-
inflammatory profile.
Due to the fact that the NAH derivative (3c) only pre-
sented anti-inflammatory activity at a high dose, that
is, 300 lmol/kg, we decided to investigate its anti-inflam-
matory profile through the administration by the alter-
native oral route. The obtained results are shown in
Figure 6. The compound (3c) (20.9% of inhibition)
had its anti-inflammatory activity reduced to half when
orally administered. This result indicates that the nitro-
acylhydrazone derivative (3c) and eventually its struc-
tural analogues are suffering a pharmacokinetic effect,
which may contribute to reduce their bioavailability,
when administered by oral route. The stomachs of the
animals used in the CIRPE assay were qualitatively
examined and no gastric irritation was observed for all
compounds even at the dose of 300 lmol/kg.39
The investigation on further SAR and the complete
mechanism of action of LASSBio-891 is now in pro-
gress. The present results highlighted the importance
of NAH-moiety as an easily prepared biophore repre-
senting a very useful privileged structure.
4. Experimental protocols
4.1. Chemistry
Melting points were determined with a Quimis 340
apparatus and are uncorrected. H NMR spectra were
1
determined in deuterated chloroform or dimethylsulfox-
ide containing ca. 1% tetramethylsilane as an internal
Figure 6. Comparison of the anti-inflammatory profile of the NBNAH
derivative (3c) administered by oral versus intra-peritoneal routes. The
test compound was administered at a dose of 300 lmol/kg. Results are
expressed as means SEM for n = 10 animals. % of inhibition was
obtained by comparison with vehicle control group (data not shown).
*p < 0.05 (Student’s t-test).
Figure 7. Antipyretic effects of the NBNAH derivatives (3a), (3c) and
dipyrone on LPS-induced fever. All compounds were administered p.o.
at a dose of 300 lmol/kg. Results are expressed as means SEM of the
differences (D) of rectal temperature for n = 8 animals. *p < 0.05 when
compared with the (D) rectal temperature at the third hour (Student’s
t-test and ANOVA).

H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
7931
standard, with Brucker DPX-200 and Varian Gemini
XI-200 spectrometers, operating at 200 MHz. 13C
NMR spectra were determined in the same spectrome-
ters described above at 50 MHz, employing the same
solvents. Microanalyses were obtained with Perkin
Elmer 240 analyzer, using Perkin Elmer AD-4 balance.
(85%) of the desired ester derivative 10, as a beige crys-
talline solid. Mp 60–62 ꢂC. 1H NMR (CDCl3) d: 6.70 (d,
Ar-H7, J = 8.4 Hz), 6.54 (d, Ar-H4, J = 2.5 Hz), 6.32
3
4
3
4
(dd, Ar-H6, J = 8.4 Hz, J = 2.5 Hz), 5.92 (s, O–CH2–
O), 4.54 (s, O–CH2C@O), 4.27 (q, O–CH2CH3,
J = 6.8 Hz), 1.30 (t, O–CH2CH3, J = 6.8 Hz); 13C
NMR (CDCl3) d: 169.1 (C@O), 153.5 (C5), 148.5 (C1),
142.6 (C3), 108.0 (C7), 106.2 (C6), 101.4 (OCH2O),
98.7 (C4), 66.7 (O–CH2C@O), 61.4 (O–CH2CH3), 14.3
(O–CH2CH3); IR (KBr) cmꢀ1: 1754 (mC@O), 1194
(mC–O).
IR spectra were obtained with BOMEM FTLA 2000-
100 spectrophotometer by using potassium bromide pel-
lets. UV spectra were determined in methanol (TEDIA)
solution on a Shimadzu UV 1601 spectrophotometer.
HPLC analyses were performed on a Shimadzu LC10
AD apparatus, with photodiode detector (SPD-M10A)
at 270 and 250 nm, equipped with a Shimadzu RP-C18
(250 · 4 mm) column. Analysis was done in gradient
mode, using a mixture of acetonitrile/water as mobile
phase at flow rate of 1 mL/min.
4.1.3. Ethyl [(6-nitro-benzo[1,3]dioxol-5-yl)oxy]acetate
(11). To a solution of ethyl ester derivative 10 (0.5 g,
2.23 mmol) in 30 mL of chloroform, cooled at 0 ꢂC,
was added dropwise 2.85 mL of concentrated HNO3
(d = 1.41 g/mL) (20 equiv). After stirring for 3 h at room
temperature, the end of the reaction was detected by
TLC analysis and then, a sufficient amount of a saturat-
ed Na2CO3 solution (ca. 15 mL) was slowly added to
neutralize the media. The organic phase was separated,
concentrated at reduced pressure, and the obtained res-
idue was washed with ice-cold water. The resulting pre-
cipitate was collected by filtration to give 0.54 g (90%) of
desired ester derivative 11, as a yellow solid, mp 85–
The progress of all reactions was monitored by TLC,
which was performed on 2.0 · 6.0 cm aluminum sheets
precoated with silica gel 60 (HF-254, Merck) to a thick-
ness of 0.25 mm. The developed chromatograms were
viewed under ultraviolet light (254–265 nm) and treated
with iodine vapor. For column chromatography Merck
silica gel (70–230 mesh) was used. Reagents and solvents
were purchased from commercial suppliers and used as
received. The usual work-up means that the organic ex-
tracts, prior to concentration under reduced pressure,
were treated with a saturated aqueous sodium chloride
solution, referred as brine, dried over anhydrous sodium
or magnesium sulfate, and filtered.
1
87 ꢂC. H NMR (CDCl3) d: 7.42 (s, Ar-H7), 6.60 (s,
Ar-H4), 6.08 (s, O–CH2–O), 4.72 (s, O–CH2C@O),
4.26 (q, O–CH2CH3, J = 7.0 Hz), 1.29 (t, O–CH2CH3,
J = 7.0 Hz); 13C NMR (CDCl3) d: 167.6 (C@O), 152.4
(C5), 149.5 (C1), 141.8 (C3), 133.3 (C6), 102.8
(OCH2O), 105.2 (C7), 97.7 (C4), 67.6 (O–CH2C@O),
61.34 (O–CH2CH3), 13.73 (O–CH2CH3); IR (KBr)
cmꢀ1: 1726 (mC@O), 1622 (mass N–O), 1193 (m C–O),
858 (mC–N).
4.1.1. Benzo[1,3]dioxol-5-ol (sesamol) (9). To a magneti-
cally stirred solution of piperonal (8) (10 g, 66.6 mmol)
in 330 mL of dichloromethane were added 18 mL of
30% aq H2O2 (166.5 mmol, 2.5 equiv) and 14 mL of for-
mic acid (4.0 equiv). The reaction mixture was main-
tained under reflux for 18 h. After cooling, 350 mL of
1.5 N NaOH solution (7.9 equiv) was added and the
mixture was stirred for additional 15 min. The aqueous
layer was additionally extracted with dichloromethane
(4· 200 mL) to remove neutral compounds. Next, the
organic layer was discarded and the remaining aqueous
phase had its pH adjusted to 1–2 with concentrated HCl
and extracted with dichloromethane (5· 200 mL). The
organic layer was separated, dried over anhydrous mag-
nesium sulfate, filtered, and vacuum concentrated,
furnishing 8.64 g of a crude pink residue. Microdistilla-
tion of this residue yielded 7.08 g (77%) of pure sesamol
(9) as a yellowish crystalline solid. Mp 64–66 ꢂC
[lit.41 mp 67 ꢂC].
4.1.4. 2-[(6-Nitro-benzo[1,3]dioxol-5-yl)oxy]acetylhydraz-
ide (12). A solution of 0.2 g of the ethyl ester derivative
11 (0.74 mmol) and 2.96 mL of 80% aq hydrazine mono-
hydrate (7.4 mmol) in 20 mL of ethanol was stirred at
room temperature for 5 min. The reaction mixture was
immediately poured into a mixture of crushed ice-water
giving a crude precipitate, which was collected by filtra-
tion and recrystallized in an ethanol/water to give 0.16 g
(85%) of hydrazide derivative 12, as a light yellow amor-
1
phous solid, mp 198–200 ꢂC. H NMR (DMSO-d6) d:
9.06 (s, CONHNH2), 7.55 (s, Ar-H7), 7.01 (s, Ar-H4),
6.17 (s, O–CH2–O), 4.66 (s, O–CH2C@O), 4.38 (s, CON-
HNH2); 13C NMR (DMSO-d6) d: 166.3 (C@O), 153.2
(C5), 150.2 (C1), 141.6 (C3), 141.6 (C6), 105.4 (C7),
103.7 (OCH2O), 97.8 (C4), 68.5 (O–CH2C@O); IR
(KBr) cmꢀ1: 3415 (mass N–H), 3383 (ms N–H), 3340 (mas
O@CN–H), 3126 (ms O@CN–H), 1667 (m C@O), 1625
(mass N–O), 1195 (m C–O).
4.1.2. Ethyl (benzo[1,3]dioxol-5-yloxy)acetate (10). A
suspension of sesamol (9) (3.34 g, 24.2 mmol) in 40 mL
of freshly distilled DMF containing anhydrous potassi-
um carbonate (10.0 g, 3.0 equiv) was stirred for 30 min
at room temperature, followed by addition of ethyl
2-bromoacetate (3.25 mL, 1.0 equiv). The reaction mix-
ture was stirred for 20 h, when the TLC analysis permit-
ted us to evidence the total consumption of 9. Next, the
reaction mixture was poured into a 1:1 mixture of
crushed ice and water (50 mL) and the resulting precip-
itate was filtered under reduced pressure, to furnish 4.6 g
4.1.5. General procedure for preparation of the 2-[(6-
nitro-1,3-benzodioxol-5-yl)oxy]acetylhydrazones (3a–h).
To a solution of 0.3 g (1.18 mmol) of hydrazide 12 in
absolute ethanol (35 mL) containing two drops of 37%
hydrochloric acid (d = 1.19 g/mL) was added ca.
1.24 mmol of the desired aromatic aldehyde, previously
dissolved in ca. 5 mL of ethanol. The reaction mixture
was stirred at room temperature for ca. 30 min, furnish-
ing an extensive precipitation. Next, the solvent was

7932
H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
0
0
concentrated at reduced pressure and the resulting resi-
due poured into ice-cold water. After neutralization with
10% aq sodium bicarbonate solution, the precipitate
formed was filtered out and dried under vacuum to give
desired N-acylhydrazone derivatives (3a–h), generally as
a colored solid.
Ar-H2 and Ar-H3 ), 7.06 (s, Ar-H4), 6.18/6.15 (s, O–
CH2–O), 5.33/4.83 (s, O–CH2C@O), 2.5 (s, -SCH3);
13C NMR (DMSO-d6) d: 168.7/163.8 (C@O), 153.3/
153.0 (C5), 150.9/150.3 (C1), 148.2 (C3), 144.2 (C@N),
0
0
141.9/141.4 (C6), 130.9 ðC4 Þ, 126.1 ðC1 Þ, 128.2/128.0
0
0
ðC2 Þ, 131.8/131.7 ðC3 Þ, 105.5/105.3 (C7), 103.9/103.6
(OCH2O), 98.1/97.9 (C4), 68.8/67.4 (O–CH2C@O), 14.8
4.1.5.1.
(40-Dimethylaminobenzylidene)-2-(6-nitro-
(–SCH3);
IR
(KBr)
cmꢀ1
:
3301
(mass
benzo[1,3]dioxol-5-yloxy)-acetylhydrazine (3a). The
derivative 3a was obtained in 98% yield, by condensa-
tion of 12 with 4-dimethylaminobenzaldehyde, as a light
–NH–), 3123 (ms –NH–), 1716 (m C@O), 1498 and 1324
(mass N–O), 1263 and 1028 (m C–O).
1
orange solid, mp 222–224 ꢂC. H NMR (DMSO-d6) d:
4.1.5.5. (10-Phenylethylidene)-2-(6-nitro-benzo[1,3]di-
oxol-5-yloxy)-acetylhydrazine (3e). The derivative 3e
was obtained in 62% yield, by condensation of 12 with
acetophenone, as a light yellow solid, mp 198–200 ꢂC.
1H NMR (DMSO-d6) d: 10.89/10.35 (s, CONH–),
11.37/11.14 (s, CONH–), 8.09/7.86 (s, –N@CH), 6.70–
0
7.59 (m, Ar-H7), 6.70–7.59 (m, Ar-H2 ), 6.70–7.59 (m,
0
Ar-H3 ), 7.07/7.04 (s, Ar-H4), 6.19/6.16 (s, O–CH2–O),
5.30/4.80 (s, O–CH2C@O), 2.96 (s, Ar–NCH3); 13C
NMR (DMSO-d6) d: 167.6/162.5 (C@O), 152.7/152.4
(C5), 151.6/151.4 (C1), 150.4/149.8 (C3), 148.7 (C@N),
0
0
0
7.21–7.89 (m, Ar-H7, Ar-H2 , Ar-H3 and Ar-H4 ), 7.13/
7.04 (s, Ar-H4), 6.19/6.15 (s, O–CH2–O), 5.37/4.92 (s,
O–CH2C@O), 2.33/2.25 (s, –CH3); 13C NMR (DMSO-
d6) d: 169.6/163.8 (C@O), 153.6/153.5 (C@N), 152.9
0
0
141.2/140.7 (C6), 121.2/121.0 ðC1 Þ, 132.1 ðC4 Þ, 128.5/
0
0
128.2 ðC2 Þ, 111.7 ðC3 Þ 104.9/104.7 (C7), 103.3/103.0
(OCH2O), 97.4/97.2 (C4), 68.26/66.7 (O–CH2C@O),
39.7 (–NCH3); IR (KBr) cmꢀ1: 3331 (mass –NH–), 3146
(ms –NH–), 1684 (m C@O), 1507 and 1324 (mass N–O),
1266 and 1033 (m C–O).
0
(C5), 150.8/150.3 (C1), 141.7/141.2 (C6), 133.2 ðC1 Þ,
0
0
0
132.7/132.1 ðC4 Þ, 128.8 ðC2 Þ, 129.9/129.6 ðC3 Þ, 105.5/
105.2 (C7), 103.8/103.5 (OCH2O), 97.7/97.6 (C4), 68.5/
67.7 (O–CH2C@O), 14.0 (–CH3); IR (KBr) cmꢀ1: 3354
(mass –NH–), 3125 (ms –NH–), 1709 (m C@O), 1510 and
1325 (mass N–O), 1266 and 1024 (m C–O).
4.1.5.2. (4-Bromobenzylidene)-2-(6-nitro-benzo[1,3]di-
oxol-5-yloxy)-acetylhydrazine (3b). The derivative 3b
was obtained in 95% yield, by condensation of 12 with
4-bromobenzaldehyde, as a light yellow solid, mp 238–
240 ꢂC. 1H NMR (DMSO-d6) d: 11.71/11.54 (s,
CONH–), 8.23/7.97 (s, –N@CH), 7.54–7.70 (m, Ar-H7,
4.1.5.6. (40-Methoxybenzylidene)-2-(6-nitro-benzo[1,3]di-
oxol-5-yloxy)-acetylhydrazine (3f). The derivative 3f was
obtained in 98% yield, by condensation of 12 with 4-
methoxybenzaldehyde, as a light yellow solid, mp 238–
240 ꢂC. 1H NMR (DMSO-d6) d: 11.46 (s, CONH–),
0
0
Ar-H2 and Ar-H3 ), 7.09 (s, Ar-H4), 6.19/6.16 (s, O–
CH2–O), 5.35/4.85 (s, O–CH2C@O); 13C NMR
(DMSO-d6) d: 168.3/163.4 (C@O), 152.4 (C5), 150.2/
149.6 (C1), 146.7 (C@N), 142.7 (C3), 141.2/140.7 (C6),
0
8.18/7.94 (s, –N@CH), 6.96–7.66 (m, Ar-H7, Ar-H2 ,
0
Ar-H3 and Ar-H4), 6.17/6.14 (s, O–CH2–O), 5.30/4.81
(s, O–CH2C@O), 3.79 (s, –OCH3); 13C NMR (DMSO-
0
0
0
0
133.2 ðC1 Þ, 123.5 /123.1 ðC4 Þ, 129.0/128.0 ðC2 Þ, 131.8/
d6) d: 168.4/163.5 (C@O), 161.5/161.2 ðC4 Þ, 153.2/
0
131.7 ðC3 Þ, 104.9/104.7 (C7, OCH2O), 97.5/97.3 (C4),
152.9 (C5), 150.8 (C1), 150.2 (C3), 144.4 (C@N), 141.2
68.1/66.7 (O–CH2C@O); IR (KBr) cmꢀ1: 3318 (mass
–
(C6), 129.3/129.0 ðC1 Þ, 127.0/126.9 ðC2 Þ, 105.4/105.2
0
0
NH–), 3186 (ms –NH–), 1682 (m C@O), 1523 and 1329
(mass N–O), 1259 and 1036 (m C–O).
(C7), 103.5 (OCH2O), 97.8 (C4), 67.2 (O–CH2C@O),
55.7 (–OCH3); IR (KBr) cmꢀ1
:
3444 (mass
–NH–), 3188 (ms –NH–), 1682 (m C@O), 1505 and 1329
4.1.5.3. Benzylidene-2-(6-nitro-benzo[1,3]dioxol-5-yl-
oxy)-acetylhydrazine (3c). The derivative 3c was ob-
tained in 98% yield, by condensation of 12 with
benzaldehyde, as a light yellow solid, mp 220–222 ꢂC.
1H NMR (DMSO-d6) d: 11.63/11.47 (s, CONH–),
(mass N–O), 1255 and 1034 (m C–O).
4.1.5.7. (30,50-Di-tert-butyl-40-hydroxybenzylidene)-2-
(6-nitro-benzo[1,3]dioxol-5-yloxy)-acetylhydrazine (3g).
The derivative 3g was obtained in 98% yield, by conden-
sation of 12 with 30,50-di-tert-butyl-40-hydroxybenzalde-
0
8.25/8.00 (s, –N@CH), 7.21–7.89 (m, Ar-H7, Ar-H2 ,
1
0
0
Ar-H3 and Ar-H4 ), 7.04 (s, Ar-H4), 6.18/6.15 (s, O–
CH2–O), 5.34/4.84 (s, O–CH2C@O); 13C NMR
(DMSO-d6) d: 168.8 (C@O), 153.0 (C5), 150.8 (C1),
hyde, as a light yellow solid, mp 248–250 ꢂC. H NMR
(DMSO-d6) d: 11.47/11.19 (s, CONH–), 8.16/7.90 (s,
0
–N@CH), 7.43–7.59 (m, Ar-H7 and Ar-H2 ), 7.08/7.02
(Ar-H4), 6.18/6.15 (s, O–CH2–O), 5.30/4.82 (s, O–
0
0
144.6 (C@N), 141.4 (C6), 134.5 ðC1 Þ, 130.6 ðC4 Þ,
0
0
129.3 ðC2 Þ, 127.8 ðC3 Þ, 105.3 (C7), 103.6 (OCH2O),
97.9 (C4), 67.4 (O–CH2C@O), 39.7 (–NCH3); IR
(KBr) cmꢀ1: 3449 (mass –NH–), 3186 (ms –NH-), 1684
(m C@O), 1519 and 1328 (mass N–O), 1143 and 1035
(m C–O).
CH2C@O), 1.39 (s, –C(CH3)3); 13C NMR (DMSO-d6)
0
d: 168.3/163.3 (C@O), 156.8/156.4 ðC4 Þ, 153.2/152.9
(C5), 150.8 (C1), 150.2/149.8 (C3), 145.6 (C@N), 141.7/
0
0
141.2 (C6), 132.6 ðC1 Þ, 125.7/125.6 ðC2 Þ, 105.4/105.2
(C7), 103.7/103.5 (OCH2O), 98.0/97.6 (C4), 68.7/68.2
(O–CH2C@O), 34.9 (–C(CH3)3); IR (KBr) cmꢀ1: 3355
(mass –NH–), 3129 (ms –NH–), 1705 (m C@O), 1515 and
1322 (mass N–O), 1269 and 1037 (m C–O).
4.1.5.4. (40-Methylthiobenzylidene)-2-(6-nitro-benzo-
[1,3]dioxol-5-yloxy)-acetylhydrazine (3d). The derivative
3d was obtained in 92% yield, by condensation of 12
with 4-(methylthio)benzaldehyde, as a dark yellow solid,
4.1.5.8. (40-Nitrobenzylidene)-2-(6-nitro-benzo[1,3]di-
oxol-5-yloxy)-acetylhydrazine (3h). The derivative 3h
was obtained in 75% yield, by condensation of 12 with
1
mp 210–212 ꢂC. H NMR (DMSO-d6) d: 11.59/11.40 (s,
CONH–), 8.19/7.95 (s, –N@CH), 7.27–7.65 (m, Ar-H7,

H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
7933
4-nitrobenzaldehyde, as a dark yellow solid, mp 242–
4.1.6. 5-(Benzyloxy)-1,3-benzodioxole (6). A suspension
of sesamol (9) (3.34 g, 24.2 mmol) in 40 mL of freshly
distilled DMF containing anhydrous potassium car-
bonate (10.0 g, 3.0 equiv) was stirred for 30 min at
room temperature, followed by addition of benzyl
bromide (2.9 mL, 1 equiv). The reaction mixture was
then stirred for 20 h, when the TLC analysis permitted
us to evidence the total consumption of 9. Next, the
reaction mixture was poured into a 1:1 mixture of
crushed ice and water (50 mL). The resulting precipi-
tate was filtered under reduced pressure to furnish
4.6 g (95%) of the desired ester derivative 6, as a beige
crystalline solid. Mp 52–54 ꢂC. 1H NMR (CDCl3) d:
244 ꢂC. 1H NMR (DMSO-d6) d: 11.91/11.60 (s,
0
CONH–), 8.36/8.09 (s, –N@CH), 8.26 (d, Ar-H2 ,
4
4
0
J = 8.2 Hz), 7.97 (d, Ar-H3 , J = 8.2 Hz), 7.58/7.52 (s,
Ar-H7), 7.10–7.08 (s, Ar-H4), 6.18/6.15 (s, O–CH2–O),
5.39/4.88 (s, O–CH2C@O); 13C NMR (DMSO-d6) d:
169.2 (C@O), 152.9 (C5), 150.6 (C1), 148.2 (C@N),
0
0
142.1 (C3), 141.3 ðC4 Þ, 140.7 ðC1 Þ, 132.8 (C6), 128.6/
0
128.4 ðC2 Þ, 105.2 (C7), 103.6 (OCH2O), 97.8 (C4), 67.3
(O–CH2C@O); IR (KBr) cmꢀ1: 3298 (mass –NH–), 1705
(m C@O), 1508 and 1342 (mass N–O), 1265 and 1035 (m
C–O).
(40-Methylsulfonylbenzylidene)-2-(6-nitro-
7.30–7.45 (m, Ar-H2 , Ar-H3 and Ar-H4 ), 6.69 (d,
Ar-H7, 3J = 8.4 Hz), 6.56 (d, Ar-H4, 4J = 2.5 Hz),
6.39 (dd, Ar-H6, 3J = 8.4 Hz, 4J = 2.5 Hz), 5.89 (s,
O–CH2–O), 4.98 (s, O–CH2Ph); 13C NMR (CDCl3)
0
0
0
4.1.5.9.
benzo[1,3]dioxol-5-yloxy)-acetylhydrazine (3i). To a solu-
tion of 0.3 g (0.77 mmol) of the nitro-acylhydrazone
derivative 3d in 30 mL of methanol were added 0.84 g
neutral alumina (Merk, Germany) and 1.66 g
(2.69 mmol) of Oxoneꢁ (Aldrich Co, USA). The result-
ing suspension was refluxed for 8 h, when TLC analysis
indicated the total consumption of the 3d. Then, reac-
tion mixture was concentrated at reduced pressure and
the obtained crude residue was submitted to silica gel
flash column chromatography eluted with dichloro-
0
d: 154.1 (C5), 148.1 (C1), 141.6 (C3), 127.7 ðC4 Þ,
0
0
0
136.9 ðC1 Þ, 127.2 ðC2 Þ, 128.3 ðC3 Þ, 107.7 (C7), 106.0
(C6), 101.9 (OCH2O), 98.3 (C4), 70.8 (O–CH2Ph); IR
(KBr) cmꢀ1: 1183 (m C–O).
4.1.7. 5-(Benzyloxy)-6-nitro-1,3-benzodioxole (5). To a
solution of benzyl ether derivative 6 (0.5 g, 2.23 mmol)
in 30 mL of chloroform, cooled at 0 ꢂC, was added
dropwise 2.85 mL of concentrated HNO3 (d = 1.41 g/
mL) (20 equiv). After stirring for 3 h, the end of the
reaction was detected by TLC analysis and a sufficient
amount of a saturated Na2CO3 solution (ca. 15 mL)
was added. The organic phase was separated, concen-
trated at reduced pressure, and the obtained residue
was washed with ice-cold water. The resulting precipi-
tate was collected by filtration to give 0.45 g (75%) of de-
sired nitroether derivative 5, as a beige solid, mp 108–
1
methane to give 3i in 85% yield, mp 184–186 ꢂC. H
NMR (DMSO-d6) d: 11.68 (s, CONH–), 7.61–7.95 (m,
0
0
–N@CH, Ar-H2 and Ar-H3 ), 7.57/7.51 (s, Ar-H7),
7.20/7.07 (s, Ar-H4), 6.18/6.15 (s, O–CH2–O), 5.35/4.85
(s, O–CH2C@O), 3.23/3.21 (s, –SO2CH3); 13C NMR
(DMSO-d6) d: 173.8/165.0 (C@O), 153.3 (C5), 149.3
0
0
(C1), 141.9 (C@N), 141.5 (C3), 141.0 ðC4 Þ, 139.1 ðC1 Þ,
0
0
132.8 (C6), 128.4/128.1 ðC2 Þ, 125.2/124.6 ðC3 Þ, 106.4
(C7), 104.8 (OCH2O), 103.5 (C4), 61.4/60.7 (O–
CH2C@O), 43.9 (–SO2CH3); IR (KBr) cmꢀ1: 3299 (mass
–NH–), 1676 (m C@O), 1509 and 1323 (mass N–O), 1311
and 1147(m SO2), 1266 and 1028 (m C–O).
1
110 ꢂC. H NMR (DMSO-d6) d: 7.54 (s, Ar-H7), 7.30–
0
0
7.48 (m, Ar-H2 , Ar-H3 and Ar-H4 ), 7.18 (s, Ar-H4),
6.15 (s, O–CH2–O), 4.98 (s, O–CH2Ph); 13C NMR
(DMSO-d6) d: 167.6 (C@O), 153.2 (C5), 150.1 (C1),
4.1.5.10.
(40-Methylsulfinylbenzylidene)-2-(6-nitro-
0
0
benzo[1,3]dioxol-5-yloxy)-acetylhydrazine (3j). To a solu-
tion of 0.3 g (0.77 mmol) of the nitroacylhydrazone
derivative 3d in 8 mL of dichloromethane was carefully
added 0.96 mmol of 3-chloroperoxybenzoic acid and the
resulting mixture was stirred at 0 ꢂC, for 4 h. Thereafter,
the reaction mixture was neutralized with a saturated
sodium bicarbonate solution and extracted with dichlo-
romethane (3· 50 mL). The collected organic layers
were dried over anhydrous sodium sulfate and concen-
trated at reduced pressure. The resulting crude residue
was submitted to silica gel flash column chromatogra-
phy eluted with dichloromethane to furnish the desired
oxidized derivative 3j in 80% yield, as a yellow solid,
mp 230–232 ꢂC. 1H NMR (DMSO-d6) d: 11.84/11.74
141.2 (C3), 136.5 ðC1 Þ, 133.3 (C6), 128.9 ðC3 Þ, 128.5
0
0
ðC4 Þ, 127.8 ðC2 Þ, 105.4 (C7), 103.6 (OCH2O), 97.8
(C4), 71.8 (O–CH2Ph); IR (KBr) cmꢀ1: 1507 and 1331
(mas N–O), 1194 (m C–O).
4.1.8. 2-(1,3-Benzodioxol-5-yloxy)acetylhydrazide (13).
A solution of 0.2 g of the ethyl ester derivative 10
(0.74 mmol) and 2.96 mL of 80% aq hydrazine mono-
hydrate (7.4 mmol) in 20 mL of ethanol was stirred at
room temperature for 5 min. The reaction mixture was
then poured into a mixture of crushed ice-water giving
a crude precipitate, which was collected by filtration
and recrystallized in an ethanol/water mixture, result-
ing in 0.12 g (80%) of hydrazide derivative 13, as a
1
0
(s, CONH–), 7.70–8.31 (m, –N@CH, Ar-H2 and
white solid, mp 198–200 ꢂC. H NMR (DMSO-d6) d:
Ar-H3 ), 7.57/7.52 (s, Ar-H7), 7.08 (s, Ar-H4), 6.18/6.15
9.28 (s, CONHNH2), 6.80 (d, Ar-H7, 3J = 8.4 Hz),
6.66 (d, Ar-H4, 4J = 2.4 Hz), 6.38 (dd, Ar-H6,
3J = 8.4 Hz and 4J = 2.4 Hz), 5.95 (s, O–CH2–O),
4.39 (s, O–CH2C@O), 4.32 (s, CONHNH2); 13C
NMR (DMSO-d6) d: 167.1 (C@O), 153.6 (C5), 148.3
(C1), 142.0 (C3), 108.4 (C6), 106.5 (C7), 101.5
(OCH2O), 98.6 (C4), 67.6 (O–CH2Ph); IR (KBr)
cmꢀ1: 3320 (mass N–H), 3240 (ms N–H), 3212 (mas
O@CN–H), 3062 (ms O@CN–H), 1678 (m C@O),
1195 (m C–O).
0
(s, O–CH2–O), 5.36/4.86 (s, O–CH2C@O), 2.76 (s,
–SOCH3); 13C NMR (DMSO-d6) d: 169.2/169.0
(C@O), 153.0 (C40), 150.8 (C5), 148.3 (C1), 141.9/
141.4 (C@N), 139.3 (C3), 136.8 (C10), 132.8 (C6),
128.4/128.2 (C20), 124.6 (C30), 105.5/105.3 (C7), 103.9/
103.6 (OCH2O), 98.2/97.9 (C4), 68.9/67.4 (O–
CH2C@O), 44.0/43.0 (–SOCH3); IR (KBr) cmꢀ1: 3299
(mass –NH–), 1708 (m C@O), 1509 and 1323 (mass N–O),
1268 and 1032 (m C–O).

7934
H. J. C. Bezerra-Netto et al. / Bioorg. Med. Chem. 14 (2006) 7924–7935
4.1.9. Benzylidene 2-(benzo[1,3]dioxol-5-yloxy)-acety-
lhydrazine (7). To a solution of 0.3 g (1.43 mmol) of
hydrazide 13 in absolute ethanol (35 mL) containing
two drops of 37% hydrochloric acid (d = 1.19 g/mL)
was added 1.50 mmol of benzaldehyde, previously dis-
solved in ca. 5 mL of ethanol. The reaction mixture
was stirred at room temperature for 30 min. Next, the
solvent was concentrated at reduced pressure and the
resulting residue was poured into ice-cold water and
neutralized with 10% aq sodium bicarbonate solution,
the precipitate formed was filtered out and dried under
vacuum to furnish 0.29 g (70% yield) of the desired acy-
lhydrazone derivative (7), as a light beige solid, mp 173–
174 ꢂC. 1H NMR (DMSO-d6) d: 11.52/11.32 (s, CONH–
respectively. The paw volumes were measured using a
glass plethysmograph coupled to a peristaltic pump,
3 h after the subplantar administration of carrageenan.
The edema was calculated as the volume variation be-
tween the carrageenan-injected paw and the saline-treat-
ed paw. The anti-inflammatory activity was expressed
as % of inhibition of the edema when compared with
vehicle control group. Results are expressed as
means SEM of n animals per group. The data were
statistically analyzed by the Student’s t-test for a signif-
icance level of *p < 0.05.
At the end of the anti-inflammatory experiments, ulcero-
genic effects in rats were investigated as described earli-
er.39 Briefly, animals were euthanized and the stomachs
excised along its greater curvature for visualization of
gastric lesions with a stereomicroscope.
0
), 8.36/8.01 (s, –N@CH), 7.67–7.71 (m, Ar-H2 ), 7.42–
0
0
7.43 (m, Ar-H3 and Ar-H4 ), 6.77–6.85 (m, Ar-H7),
6.68 (d, Ar-H6, J = 2.3 Hz and J = 11.1 Hz), 6.39 (d,
4
3
4
Ar-H4, J = 2.3 Hz), 5.97/5.95 (s, O–CH2–O), 5.05/4.59
(s, O–CH2C@O); 13C NMR (DMSO-d6) d: 169.6/164.8
(C@O), 154.1/153.6 (C5), 148.5 (C@N), 148.4 (C1),
4.2.3. Antipyretic activity. The antipyretic effect was
determined in vivo by using lipopolysaccharide (LPS)-
induced fever (50 lg/kg, i.p.) in mice.40 Albino mice of
both sexes (28–32 g) were used. The rectal temperature
was measured by inserting a lubricated digital thermom-
eter, with a 0.1 ꢂC precision, 3 cm into the rectum of the
animal. Two control measurements were carried out at
1 h interval before administration of LPS. The second
recorded temperature, taken immediately before LPS
injection, was defined as the basal rectal temperature.
Three hours after LPS, the rectal temperatures were
recorded again immediately before the oral administra-
tion of the compounds (300 lmol/kg) as a suspension
of 5% Arabic gum in saline (vehicle). The rectal temper-
atures were recorded during the following 3 h. Dipyrone
(300 lmol/kg) was used as standard under the same con-
ditions. Results are expressed as means SEM of the
differences (D) between the rectal temperature at each
time and the basal one, for n animals per group. Data
were statistically analyzed by the Student’s t-test and
ANOVA (one-way) for a significance level of *p < 0.05
when compared with the (D) rectal temperature at the
third hour.
0
0
148.3 (C3), 142.2/141.8 ðC1 Þ, 134.6/134.4 ðC4 Þ, 129.2
0
0
ðC2 Þ, 127.6/127.4 ðC3 Þ, 108.5/108.4 (C7), 106.5/106.3
(C6), 101.6/101.5 (OCH2O), 98.6/98.5 (C4), 67.9/66.0
(O–CH2C@O); IR (KBr) cmꢀ1: 3093 (ms -NH-), 1681
(m C@O), 1141 and 1035 (m C–O).
4.2. Pharmacology
4.2.1. Analgesic activity. The analgesic activity was
determined in vivo by the acetic acid-induced (0.6%,
0.1 mL/10 g) abdominal constriction test in mice.37 Albi-
no mice of both sexes (18–23 g) were used. Compounds
were administered orally (100 lmol/kg) as a suspension
in 5% Arabic gum in saline (vehicle). Dipyrone
(100 lmol/kg) and nimesulide (1) (100 lmol/kg) were
used as standard drugs under the same conditions. Ace-
tic acid solution was administered i.p. 1 h after adminis-
tration of the compounds. Ten minutes after the i.p.
acetic acid injection, the number of constrictions per
animal was recorded for 20 min. Control animals re-
ceived an equal volume of vehicle. Analgesic activity
was expressed as % of inhibition of constrictions when
compared with the vehicle control group. Results are
expressed as means SEM of n animals per group.
The data were statistically analyzed by the Student’s
t-test for a significance level of *p < 0.05.
Acknowledgment
Thanks are due to IM-INOFAR (BR, #420.015/05-1),
PRONEX-Rio (BR, #171.162/2003), CNPq (BR,
#302.231/2003-0, #474.521/2004-4, #470.883/2004-9,
#500.185/2003-4, #479.360/2004-9), FAPERJ (BR,
#151.939/2004, #151.509/1999, #171.129/2003), CAPES
(BR, PROCAD-2005) and FUJB (BR) for the financial
support and fellowships.
When appropriated, the ID50 values were determined by
non-linear regression using GraphPad Prism software
(Version 3.00, 1999).
4.2.2. Anti-inflammatory activity. The anti-inflammatory
activity was determined in vivo by the carrageenan-in-
duced rat paw edema test according to Ferreira.38 Fast-
ed albino rats of both sexes (150–200 g) were used. Test
compounds and the standard anti-inflammatory drug
nimesulide (1) were administered orally and intraperito-
neally (300 lmol/kg) as a suspension in 5% Arabic gum
in saline (vehicle). Control animals received an equal
volume of vehicle. On hour later, the animals were then
injected with either 0.1 mL of 1% carrageenan solution
in saline (0.1 mg/paw) or sterile saline (NaCl 0.9%)
into the subplantar surface of one of the hind paw,
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