Novel 6-methanesulfonamide-3,4-methylenedioxyphenyl-N-acylhydrazones: Orally effective anti-inflammatory drug candidates

Article abstract of DOI:10.1016/j.bmc.2008.12.045

We described herein the molecular design of novel in vivo anti-inflammatory 6-methanesulfonamide-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives (1) planned by applying the molecular hybridization approach. This work also points out to the discovery

Full text of DOI:10.1016/j.bmc.2008.12.045

Bioorganic & Medicinal Chemistry 17 (2009) 1125–1131
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 6-methanesulfonamide-3,4-methylenedioxyphenyl-N-acylhydrazones:
Orally effective anti-inflammatory drug candidates
Jorge L. M. Tributino a,b, Carolina D. Duarte a,c, Rodrigo S. Corrêa d,e, Antonio C. Doriguetto d,
Javier Ellena e, Nelilma C. Romeiro a, Newton G. Castro b, Ana Luisa P. Miranda a,
Eliezer J. Barreiro a,b,c, Carlos A. M. Fraga a,b,c,
*
a LASSBio—Laboratório de Avaliação e Síntese de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, PO Box 68023,
21941-902 Rio de Janeiro, RJ, Brazil
b Programa de Pós-Graduação em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
d Laboratório de Cristalografia, Departamento de Ciências Exatas, Universidade Federal de Alfenas—UNIFAL-MG, 37130-000 Alfenas, MG, Brazil
e Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We described herein the molecular design of novel in vivo anti-inflammatory 6-methanesulfonamide-
3,4-methylenedioxyphenyl-N-acylhydrazone derivatives (1) planned by applying the molecular
hybridization approach. This work also points out to the discovery of LASSBio-930 (1c) as a novel anti-
inflammatory and anti-hyperalgesic prototype, which was able to reduce carrageenan-induced rat paw
Received 13 September 2008
Revised 13 December 2008
Accepted 17 December 2008
Available online 25 December 2008
edema with an ED50 of 97.8
lmol/kg, acting mainly as a non-selective COX inhibitor.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
N-Acylhydrazone derivatives
Anti-inflammatory
Anti-hyperalgesic
COX-1 inhibitor
Safrole
1,3-Benzodioxole
1. Introduction
matory properties of orally active flosulide analogues derived from
natural safrole.7 More recently, Khanapure et al.8 described metha-
ryl and methacycloalkyl derivatives and ketone analogues, structur-
ally related to our flosulide analogues, as potent cycloxygenase-2
(COX-2) inhibitors, anti-inflammatory agents.
Inflammation can be defined as an intricate response from dam-
aged tissue, which consists in assembled interactions among cell-
derived mediators leading to recovery. Conversely, in pathological
conditions evolving persistent tissue damage by inflammatory
cells, repair is not suitably accomplished.1 Eicosanoids, important
mediators of the inflammatory process, are able to activate and
sensibilize nociceptors, leading to hyperalgesia2 and their role in
the genesis of several pathological states, such as chronic inflam-
matory diseases, thrombosis and pain, is well established.3
In the recent literature, we have described the structure–activity
relationships of numerous series of N-acylhydrazones (NAH), with
potent anti-inflammatory, antinociceptive and antiplatelet activi-
ties.4,5 The relative acidity of the amide hydrogen of the NAH func-
tion, as well as its capacity for stabilizing free radicals give these
compounds the ability of mimicking the bis-allylic moiety of unsat-
urated fatty acids and amides, for example, arachidonicacid (AA).6 In
addition, wehavepreviouslyreportedthesynthesisandanti-inflam-
Ourpresentwork describesthe development of novel drugcandi-
dates with an orally active anti-inflammatory and analgesic profile,
based on our previous results concerning the medicinal chemistry of
NAH and 1,3-benzodioxole-containing derivatives.4–7 Aiming to
optimize the NAH privileged scaffold9 to achieve more effective
anti-inflammatory compounds, the rational approach leading to
the new 6-methanesulfonamide-3,4-methylenedioxyphenyl-N-
acylhydrazones (1a–d) included molecular hybridization of LASS-
Bio-294 (2), a potent analgesic and antiplatelet derivative but poor
anti-inflammatory,10 and LASSBio-259 (3), a flosulide analogue with
potent in vivo anti-inflammatory properties.7 The design concept of
compounds (1a–d) explored the introduction of a methanesulfon-
amide group in the 1,3-benzodioxole ring at position 6 and the
NAH moiety working as a linker between the two peripheral aro-
matic rings (Scheme 1). The nature of the aryl groups attached to
the imine subunit of the new series of compounds (1a–d), that is
2-furyl, 2-thiophenyl and phenyl, was elected based on classical ring
* Corresponding author. Tel.: +55 21 25626503; fax: +55 21 25626644.
E-mail address: cmfraga@pharma.ufrj.br (C.A.M. Fraga).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.045

1126
J. L. M. Tributino et al. / Bioorg. Med. Chem. 17 (2009) 1125–1131
isosteric replacement11 while 3,5-di-tert-butyl-4-hydroxyphenyl
was elected aiming at introducing the characteristic redox proper-
ties of this moiety into the new series of compounds, as previously
reported for other series of bioactive NAH derivatives.5f
(4, Scheme 2)12 was used as starting material. As recently de-
scribed by our group,5f 6-nitropiperonyl methyl ester (5) was pre-
pared in 63% yield from safrole (4 steps), by means of
isomerization and oxidative cleavage,13 nitration14 and Yamada’s
oxidation conditions.15 The regioselective introduction of the nitro
group was clearly evidenced by usual spectroscopic methods as
previously described.5f,14
The nitro-ester (5) was then submitted to chemoselective nitro
group reduction with metallic iron in acidic media,7 affording the
corresponding aniline derivative (6) in 75% yield. Further mesyla-
tion reaction7 furnished the corresponding N-methanesulfonyl
derivative (7) in 89% yield. Infrared spectra of the former com-
pound shows absorption bands in 3140 and 3108 cmꢀ1 referring
to axial deformation of N–H, whilst 1675, 1362 and 1148 cmꢀ1
bands are characteristic of carbonyl group deformation and asym-
metric and symmetric deformations of the –SO2 group,
respectively.
2. Results and discussion
2.1. Chemistry
For the synthesis of the new substituted 6-methanesulfonam-
ide-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives (1),
an abundant natural product from Brazilian flora, that is, safrole
The key hydrazide intermediate (8) was then obtained in 78%
yield, by treating an ethanolic solution of (7) with hydrazine hy-
drate at room temperature for 1 h.5e Finally, subsequent condensa-
tion with the appropriate aromatic aldehydes in ethanol5e
provided the new NAH target compounds (1a–d) (Table 1), under
acid catalysis, in 22–27% overall yield from (4). The careful analysis
of 1H NMR spectra (Table 1) of the obtained NAH (1) indicated that
the condensation reaction was highly diastereoselective since only
the E-diastereomer at the imine double bond level was evidenced,
in agreement with our previous results.5
In order to assure unambiguously the relative configuration of
the diastereomer obtained during the synthesis of these new
1,3,benzodioxole NAH derivatives, essential to the understanding
of the biological results, we have selected the unsubstituted com-
pound LASSBio-930 (1c), to investigate the geometric isomerism
and other conformational factors by X-ray diffraction.
Scheme 1. Design concept for 6-methanesulfonamide-3,4-methylenedioxyphenyl-
N-acylhydrazone derivatives (1a–d).
The ORTEP-316 view of LASSBio-930 (1c) with the atom num-
bering scheme is shown in Figure 1. The main geometric param-
eters are given in Supplementary data. There are two molecular
moieties, which are individually almost flat: the moiety 1 con-
taining the atoms C8 and N3 and those ones present in rings
A and B; and the moiety 2 containing ring C and N1, N2 and
C9. The largest deviations from the least squares plane through
the moieties 1 and 2 are 0.036(2) and ꢀ0.046(2) Å for N1 and
C8 atoms, respectively. The least squares planes of the moieties
1 and 2 form an angle of 13.4(1)°. This geometric feature is also
highlighted by the torsional angle considering C6–C1–C8–N2
(torsion = 22.0(1)°). The O1 atom is slightly closer to the planar
moiety 2 (0.323(2) Å) than the planar moiety 1 (0.371(3) Å). An-
other important intra-molecular information revealed by the X-
ray crystallographic analysis is the orientation of the methane-
sulfonamide group. The S1 and O6 atoms deviate 0.229(2) and
0.122(3) Å from the least squares plane through the moiety 1,
whereas for the O5 and C22 atoms the deviations are
ꢀ0.671(3) and 1.853(3) Å, respectively.
Scheme 2. Synthesis of 1,3-benzodioxole NAH derivatives (1a–d). Reagents and
conditions: (a) Fe0, NH4Cl, EtOH/H2O, reflux, 2 h, 75%; (b) CH3SO2Cl, Py, CH2Cl2, rt,
1 h, 89%; (c) N2H4ꢁH2O 80%, EtOH, rt, 1 h, 78%; (d) ArCHO, EtOH, rt, 0.5 h, 70–88%.
Table 1
Yields and physicochemical properties of new 6-methanesulfonamide-3,4-methylenedioxyphenyl-N-acylhydrazone NAH derivatives (1a–d)
Compound
Yielda (%)
Molecular formulab
Melting pointc (°C)
Dd (ppm)
SO2NH
CONH
N@CH
1a
1b
1c
1d
86
88
84
70
C14H13N3O6S
C14H13N3O5S2
C16H15N3O5S
C24H31N3O6S
260–262
276–278
244–246
254–255
11.8
11.8
11.9
11.7
10.6
10.6
10.7
10.7
7.1
7.3
8.4
8.3
a
Isolated yield.
b
c
The analytical results for C,H,N were within 0.4% of calculated values.
Not corrected.
d
Data obtained 1H NMR at 200 MHz, using DMSO-d6 as solvent.

J. L. M. Tributino et al. / Bioorg. Med. Chem. 17 (2009) 1125–1131
1127
The compound LASSBio-930 (1c) exhibits a moderate intra-
molecular hydrogen bond involving N3–H1ꢁꢁꢁO1. The weak inter-
molecular hydrogen bond between O5 in the methanesulfonam-
ide and the adjacent hydrogen donor group N2–H2 at ꢀx,
ꢀy + 1, ꢀz helps in the packing stabilization and gives rise to
the dimer. The remaining O atom present in the methanesulfon-
amide subunit, O6, also contributes to the packing stabilization
forming bifurcated non-classical H bonds (C9–H9ꢁꢁꢁO6 and C22–
H22CꢁꢁꢁO6). Another non-classical inter-molecular hydrogen bond
occurs between C7–H7AꢁꢁꢁO2, which forms a centrosymmetrical
dimer linked by the heterocyclic five membered ring. All informa-
tion concerning the inter-molecular geometry are given in the
Supplementary data.
2.2. Pharmacology
Figure 1. ORTEP view of LASSBio-930 (1c) showing the arbitrary atom labelling.
Ellipsoids represent 50% probability level.
For the in vivo pharmacological evaluation of the new substi-
tuted 6-methanesulfonamide-3,4-methylenedioxyphenyl-N-acy-
lhydrazone derivatives (1a–d), we elected the carrageenan-
induced rat paw edema model (CIRPE), a classic model of acute
inflammation17 (Fig. 2). Nimesulide, a diarylether anti-inflamma-
tory drug and LASSBio-294 (2) were used as standards. All com-
pounds were administered orally (300
inflammatory challenge.
lmol/kg) 1 h before
The new compounds LASSBio-928 (1a), LASSBio-929 (1b) and
LASSBio-930 (1c) were more effective on rat paw edema preven-
tion than prototype LASSBio-294 (2). These results point out to
the importance of the methanesulfonamide group at C6 of the
1,3-benzodioxole ring for the improvement of anti-inflammatory
activity. However, LASSBio-931 (1d), the 3,5-di-tert-butyl-4-
hydroxyphenyl derivative, did not show a significant anti-inflam-
matory effect (Fig. 2), which may have been the result of a higher
lipophilicity and lower solubility of this compound as compared to
the other ones, which could contribute to differences in their
bioavailability.
Figure 2. Anti-inflammatory evaluation of 6-methanesulfonamide-3,4-methylene-
dioxyphenyl-N-acylhydrazone (1a–d) on carragenaan-induced rat paw edema.
Results refer to the 3rd hour paw volumes after carrageenan-challenge and are
expressed in terms of mean SEM (n = 8–10 animals per group). Compounds were
administered orally at a dose of 300 l
mol/kg. *p < 0.05 compared to the vehicle
group (Student t test).
Moreover, the most active compound on this pharmacological
protocol (ca. 60% inhibition), that is, LASSBio-930 (1c), presented
a dose-dependent profile with an ED50 = 97.8 lmol/kg and effec-
tiveness similar to the standard drug nimesulide.18 The edema
was inhibited from the 2nd hour after carrageenan injection. LASS-
Bio-930 also demonstrated an anti-inflammatory effect on the ara-
chidonic acid-induced ear edema (data not shown). For this reason,
compound (1c) can be considered as the representative hybrid
analog of LASSBio-259 (3) where the methylene spacer was substi-
tuted by the NAH moiety from LASSBio-294 (2) (Scheme 1).
Additionally, LASSBio-930 (1c) was able to reduce significantly
the associated hyperalgesia induced by carrageenan in the same
model (Fig. 3), corroborating the preliminary antinociceptive activ-
ity demonstrated by means of the inhibition of the AcOH-induced
constrictions by 70% at 300 lmol/kg and with the inhibition of the
inflammatory phase of the formalin-induced hyperalgesia test,
both in mice (Fig. 4). Carragenan-induced inflammation is charac-
terized by the production of PGE2 in tissues resulting in hyperalge-
sia. Thus, it has been demonstrated that NSAIDs like indomethacin
and nimesulide are able to inhibit paw edema production and ther-
mal hyperalgesia.19,20
In order to investigate if the anti-inflammatory profile of LASS-
Bio-930 (1c) was dependent on COX blockage, we evaluated its
ability to inhibit the isoforms 1 and 2 of this enzyme by using
the human whole blood (HWB) in vitro assay.21,22 COX-1 activity
was measured through TXB2 production in a spontaneous human
whole blood clot assay and COX-2 activity through TXB2 produc-
tion from whole blood samples stimulated by lipopolysaccharide
(LPS). The IC50 values for COX’s and the selectivity index were
determined for LASSBio-930 (1c) and nimesulide, as summarized
in Table 2.
Figure 3. Time course effect of LASSBio-930 (1c) and nimesulide on carrageenan-
induced rat paw edema (A) and thermal hyperalgesia (B). Results are expressed in
terms of mean SEM (n = 8–10 animals per group). LASSBio-930 and nimesulide
(300 l
mol/kg; p.o.). *p < 0.05 compared to the vehicle control group (Student’s t
test).

1128
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Table 3
D
Gbinding values (kJ/mol) obtained for LASSBio-930 (1c) and nimesulide through
Chemscore function in GOLD program
Compound
DGbinding
COX-1
COX-2
Nimesulide
LASSBio-930 (1c)
ꢀ31.75
ꢀ39.85
ꢀ33.27
ꢀ37.03
However, the visual inspection of the best-ranked complex of
LASSBio-930 with COX-2 (Fig. 5B) shows an inverted putative bind-
ing mode in comparison to COX-1, in which there is one hydrogen
bond between one of the oxygen atoms of the sulfonamide group
and the guanidine group of Arg120. Although these observations
suggest more favourable polar interactions of LASSBio-930 with
Figure 4. Effect of LASSBio-930 (1c) and nimesulide in mice formalin-induced pain
test. Each column represents the mean SEM (n = 8 animals per group). LASSBio-
930 and nimesulide (300 l
mol/kg; p.o). *p < 0.05 compared to the vehicle control
group (Student’s t test).
COX-2 than COX-1, which is not evidenced by the
DGbinding energy
term, Glipo values obtained for COX-1 and this compound (data
D
Table 2
not labeled) are more favourable than for COX-2 (249.10 vs
233.83 kJ/mol, respectively), suggesting more favourable hydro-
phobic interactions between LASSBio-930 and COX-1 and corrobo-
rating the low COX-1-selective profile observed experimentally for
this compound.
In vitro COX-1 and COX-2 human whole blood inhibition assay for LASSBio-930 (1c)
and nimesulide
a
Compound
HWB IC50
COX-1
(
l
M)
Selectivity index
IC
50COX-1/IC50COX-2
COX-2
Nimesulide
LASSBio-930 (1c)
10.9 1.2
4.1 1.1
3.5 1.3
10.2 1.3
3.1
0.4
3. Conclusions
a
IC50 values (i.e., the concentration able to elicit 50% of the maximum effect
observed) were determined by non-linear regression using GraphPad Prism soft-
ware. Each point of the curve represents the mean SEM of the % of inhibition of
TXB2 formation (n = 3–5 independent experiments). Concentration range for COX-1
LASSBio-930. (1c) was identified as a new anti-inflammatory
and anti-hyperalgesic prototype belonging to the methanesulfon-
amide N-acylhydrazone series, which exert their bioactivities
through non-selective COX inhibition. This compound was elected
for further pharmacological evaluation on chronic inflammation
and pain models in order to confirm its potential as a drug candi-
(0.01–500 lM). Concentration range for COX-2 (0.001–1000 lM).
The obtained results showed that LASSBio-930 (1c) presented a
COX-1 and COX-2 inhibitory profile similar to that observed for
nimesulide, as previously anticipated from our in vivo CIRPE re-
sults, with an inverse selectivity profile (SI = 0.4 and 3.1, respec-
tively) (Table 2). Additionally, in spite of the presence of the
methanesulfonamide subunit in the hybrid analogue LASSBio-930
(1c), classically known as a pharmacophoric group for COX-2 inhi-
bition,23 it has proven to be
(IC50 = 4.1 M).
a
better COX-1 inhibitor
l
2.3. Molecular docking
Aiming to support the in vitro COX inhibitory profile displayed
by LASSBio-930 (1c) and nimesulide we performed molecular
docking studies using available COX-1 and COX-2 X-ray crystal
structures with bound inhibitors.24,25 This theoretical study was
performed using the genetic algorithm-based docking program
26
GOLD
.
The proposed binding modes of the ligand into the active
site of COX-1 and COX-2 were determined as the best ranked com-
pounds according to ChemScore scoring function,27 which corre-
sponds to the structure with the most favourable free energy of
binding, that is,
DGbinding (kJ/mol) (Table 3).
The Gbinding values obtained reveal that nimesulide has a slight
D
preference for binding COX-2, with a small difference of 1.52 kJ/
mol, as it is observed experimentally. The same feature was ob-
served for LASSBio-930, except that its binding to COX-1 is ener-
getically more favourable than to COX-2 by 2.82 kJ/mol (Table 3).
These results suggest a non-selective profile for this compound,
as has been observed in the in vitro pharmacological assays, since
the energy difference is so small.
The visual analysis of the docked complexes between LASSBio-
930 and COX-1 (Fig. 5A) shows that LASSBio-930 is able to form
one hydrogen bond involving the hydrogen atom of the imine moi-
ety and the hydroxyl of Ser530.
Figure 5. Best-ranked LASSBio-930-COX-1 (A) and LASSBio-930-COX-2 (B) binding
poses as obtained by docking with ChemScore scoring function. Hydrogen bonds
are shown as green dashed lines. Hydrogen-bonding interacting residues are
labeled yellow. Only polar hydrogens are shown for clarity.

J. L. M. Tributino et al. / Bioorg. Med. Chem. 17 (2009) 1125–1131
1129
date for treatment of different inflammatory diseases and these re-
sults will be disclosed in a forthcoming paper.
the desired compound (8) as a yellow solid (0.39 g, 78%), mp 198–
200 °C. 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 3.03 (s, 3H,
SO2CH3); 4.60 (s, 2H, NH2); 6.10 (s, 2H, H-2); 7.06 (s, 1H, H-4); 7.32
(s, 1H, H-7); 9.96 (s, 1H, SO2NH); 11.29 (s, 1H, CONHNH2). 13C NMR
(50 MHz, DMSO-d6, TMS) d (ppm): 40.0 (SO2CH3); 101.8 (C-2);
102.7 (C-7); 107.2 (C-4); 113.0 (C-5); 135.4 (C-6); 143.9 (C-3a);
150.8 (C-1a); 167.4 (C@O).
4. Experimental
4.1. Chemistry
Reactions were routinely monitored by thin-layer chromatogra-
phy (TLC) in Silica Gel (F245 Merck plates) and the products visu-
4.1.4. General procedure for preparation of 6-methanesulfonam-
ide-N0-arylidene-1,3-benzodioxole-5-carbohydrazines (1a–d)5e
To a solution of 1 mmol of the hydrazide (8) in absolute ethanol
(10 mL) containing two drops of 37% hydrochloric acid, was added
1.1 mmol of corresponding aromatic aldehyde derivative. The mix-
ture was stirred at room temperature for 1 h, after then extensive
precipitation was visualized. Next, the mixture was poured into
cold water, neutralized with 10% aqueous sodium bicarbonate
solution and the precipitate formed was filtered out and recrystal-
lized from ethanol/water.
alized with iodine or ultraviolet lamp (254 and 365 nm). 1H and 13
C
nuclear magnetic resonance (NMR) spectra were determined in
DMSO-d6 solutions using a Bruker AC-200 spectrometer. Peak posi-
tions are given in parts per million (d) from tetramethylsilane 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 bro-
mide (KBr) disks. Melting points were determined using a Quimis
instrument and are uncorrected. Column chromatography purifica-
tions were performed using silica gel Merck 230–400 mesh. All or-
ganic solutions were dried over anhydrous sodium sulfate and all
organic solvents were removed under reduced pressure in rotatory
evaporator.
4.1.5. N-(6-{[(2E)-2-(Furan-2-ylmethylidene)hydrazinyl]car-
bonyl}-1,3-benzodioxol-5-yl)methanesulfonamide (LASSBio-928, 1a)
The derivative (1a) was obtained in 86% yield, as a light yellow so-
lid, mp 260–262 °C. 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 3.08
(s, 3H, SO2CH3); 6.15 (s, 2H, H-2); 6.65 (s, 1H, H-400);6.95(s, 1H, H-300);
7.05(s, 1H, H-40);7.41(s, 1H, H-4);7.85(s, 1H,H-500);8.28(s, 1H, H-7);
10.60 (s, 1H, SO2NH); 11.77 (s, 1H, H-20). 13C NMR (50 MHz, DMSO-d6,
TMS) d (ppm): 40.3 (SO2CH3); 102.9 (C-2); 108.7 (C-300); 112.7 (C-400);
114.5 (C-7); 114.9 (C-500);135.2 (C-5); 138.8 (C-40); 144.1 (C-6); 145.9
(C-4); 149.7 (C-1a); 151.0 (C-3a); 164.4 (C-10).
4.1.1. Methyl 6-amino-1,3-benzodioxole-5-carboxylate (6)7
A mixture of 1.0 g (4.40 mmol) of methyl 6-nitro-1,3-benzodi-
oxole-5-carboxylate (5), 1.39 g (24.9 mmol) of powder metallic
iron and 0.14 g (2.70 mmol) of ammonium chloride suspended in
75 mL of ethanol/water (2:1) was refluxed for 1 h, when TLC indi-
cated the end of the reaction. Then, the reaction mixture was fil-
tered over CeliteÒ and the filtrate was concentrated under
reduced pressure, furnishing a crude dark yellow solid. Recrystalli-
zation from ethanol/water, afforded the title compound as a yellow
solid (0.65 g, 75%), mp 175–176 °C. 1H NMR (200 MHz, CDCl3, TMS)
d (ppm): 3.83 (s, 3H, OCH3); 5.60 (s, 2H, NH2); 5.89 (s, 2H, H-2);
6.17 (s, 1H, H-4); 7.26 (s, 1H, H-7). 13C NMR (50 MHz, CDCl3,
TMS) d (ppm): 51.3 (OCH3); 96.8 (C-2); 101.2 (C-7); 102.5 (C-4);
108.5 (C-5); 139.1 (C-6); 148.8 (C-3a); 152.9 (C-1a); 168.2 (C@O).
4.1.6. N-(6-{[(2E)-2-(Thiophen-2-ylmethylidene)hydrazinyl]car-
bonyl}-1,3-benzodioxol-5-yl)methanesulfonamide (LASSBio-929, 1b)
The derivative (1b) was obtained in 82% as a light yellow solid,
mp 276–278 °C. 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 3.08
(s, 3H, SO2CH3); 6.14 (s, 2H, H-2); 7.12 (d, 1H, J = 4 Hz, H-400); 7.16
(d, 1H, J = 4 Hz, H-300); 7.33 (s, 1H, H-40); 7.42 (s, 1H, H-4); 7.69 (d,
1H, J = 4 Hz, H-500); 8.59 (s, 1H, H-7); 10.59 (s, 1H, SO2NH); 11.81 (s,
1H, H-20). 13C NMR (50 MHz, DMSO-d6, TMS) d (ppm): 40.2
(SO2CH3); 102.8 (C-2); 102.9 (C-300); 108.1 (C-400); 114.9 (C-5);
128.4 (C-7); 129.8 (C-500); 131.9 (C-40); 135.2 (C-6); 139.3 (C-1a);
144.1 (C-4); 151.0 (C-3a); 164.3 (C-10).
4.1.2. Methyl 6-[(methylsulfonyl)amino]-1,3-benzodioxole-5-
carboxylate (7)7
To a solution of 0.5 g (2.60 mmol) of (6) in 30 mL of dichorome-
thane containing 3.0 mL of anhydrous pyridine, was added 0.9 mL
(11.60 mmol) of freshly distilled mesyl chloride. The reaction mix-
ture was kept in room temperature for 1 h, when TLC indicated the
end of the reaction. Then, the organic media was sequentially
washed with 10% aq HCl (3 ꢂ 30 mL), saturated CuSO4 solution
(2 ꢂ 30 mL) and water (2 ꢂ 30 mL), dried and concentrated under
reduced pressure to furnish (7) as a white solid (0.62 g, 89%), mp
178–180 °C. 1H NMR (200 MHz, CDCl3, TMS) d (ppm): 3.02 (s, 3H,
SO2CH3); 3.91 (s, 3H, OCH3); 6.05 (s, 2H, H-2); 7.31 (s, 1H, H-4);
7.44 (s, 1H, H-7); 10.00 (s, 1H, NH). 13C NMR (50 MHz, CDCl3, TMS)
d (ppm): 40.0 (SO2CH3); 52.6 (OCH3); 100.6 (C-2); 102.5 (C-7);
108.9 (C-4); 109.6 (C-5); 138.2 (C-6); 143.8 (C-3a); 153.2 (C-1a);
4.1.7. N-(6-{[(2E)-2-Benzylidenehydrazinyl]carbonyl}-1,3-
benzodioxol-5-yl)methanesulfonamide (LASSBio-930, 1c)
The derivative (1c) wasobtained in95%, asa yellowsolid, mp244–
246. 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 3.09 (s, 3H, –
SO2CH3); 6.15 (s, 2H, H-2); 7.12 (s, 1H, H-4); 7.46 (m, 4H, H-
200,300,500,600); 7.73 (m, 2H, H-7,400); 8.41 (s, 1H, H-40); 10.66 (s, 1H, –
SO2NH); 11.87 (s, 1H, H-20). 13C NMR (50 MHz, DMSO-d6, TMS) d
(ppm): 40.3 (–SO2CH3); 102.8 (C-7); 102.9 (C-2); 108.1 (C-4); 114.8
(C-5); 127.7 (C-200,600); 129.4 (C-300,500); 130.8 (C-4”); 134.6 (C-100);
135.4 (C-6); 144.0 (C-3a); 149.1 (C-40); 151.1 (C-1a); 164.5 (C-10).
168.2 (C@O). IR (KBr)
m
(cmꢀ1): 3140, 3108, 3017, 2952, 2932,
4.1.8. N-(6-{[(2E)-2-(4-Hydroxy-3,5-di-tert-butyl-benzylidene)
hydrazinyl]carbonyl}-1,3-benzodioxol-5-yl)methanesulfonamide
(LASSBio-931, 1d)
2852, 1675, 1627, 1507, 1486, 1443, 1362, 1332, 1244, 1148, 1030,
978, 903, 880, 784, 647, 500.
The derivative (1d) was obtained in 70% yield, as a yellow solid,
mp 254–255. 1H NMR (200 MHz, DMSO-d6, TMS) d (ppm): 1.41 (s,
18H, –C(CH3)3); 3.08 (s, 3H, –SO2CH3); 6.14 (s, 2H, H-2); 7.11 (s,
1H, –OH); 7.46 (m, 4H, H-4,7,200,600); 8.32 (s, 1H, H-40); 10.73 (s, 1H,
–SO2NH); 11.67 (s, 1H, H-20). 13C NMR (50 MHz, DMSO-d6, TMS) d
(ppm): 33.7 (–C(CH3)3); 38.1 (–SO2CH3); 43.3 (–C(CH3)3); 105.8 (C-
7); 106.0 (C-2); 111.2 (C-4); 117.9 (C-5); 127.7 (C-200,600); 128.9 (C-
300,500); 138.4 (C-100); 142.8 (C-6); 147.1 (C-1a); 153.7 (C-40); 154.0
(C-3a); 160.0 (C-400); 167.3 (C-10).
4.1.3. N-[6-(hydrazinylcarbonyl)-1,3-benzodioxol-5-
yl]methanesulfonamide (8)5e
To a solution of 0.5 g (1.80 mmol) of (7) in 10 mL of ethanol, was
added 5.8 mL (92.8 mmol) of 80% hydrazine monohydrate. The reac-
tion mixture was kept in room temperature for 1 h, when TLC indi-
cated the end of the reaction. Then, the media was neutralized
with concentrated HCl in an ice bath and the resulting precipitate
was filtered out and recrystallized from ethanol/water, affording

1130
J. L. M. Tributino et al. / Bioorg. Med. Chem. 17 (2009) 1125–1131
Table 4
4.2. Single crystal X-ray diffraction
Crystal data and structure refinement for NAH derivative LASSBio-930 (1c)
After the synthesis and purification procedures, a well-shaped
clear single crystal of (LASSBio-930), which was grown by recrystal-
lization from 1:1 dichloromethane and methanol mixture, was se-
lected for the X-ray diffraction experiment. Intensity data were
measured with the crystal at room temperature (298 K) and with
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C16H15N3O5S1
361.37
298(2)
0.71073
Monoclinic
P21/c
graphite monochromated Mo Ka radiation (k = 0.71073 Å), using
11.2724(4)
8.4786(2)
17.1899(5)
the Enraf-Nonius Kappa-CCD diffractometer. The cell refinements
b (Å)
c (Å)
28
29
were performed using the software COLLECT
and SCALEPACK
,
and
thefinalcellparameterswereobtainedonallreflections. Datareduc-
tion was carried out using the software DENZO-SMN and SCALEPACK. Since
the absorption coefficient is insignificant (0.232 mmꢀ1), no absorp-
tion correction was applied.
a
b (°)
(°)
90
97.463(1)
90
1628.99(8)
4
1.473
0.232
c
(°)
Volume (Å3)
Z
The structure was solved using the software SHELXS-9730 andrefined
using the software SHELXL-97.31 Non-hydrogen atoms of the molecules
were clearly solved and full-matrix least-squares refinement of these
atoms with anisotropic thermal parameters was carried on. The C–H
hydrogen atoms were positioned stereochemically and were refined
with fixed individual displacement parameters [Uiso(H) = 1.2Ueq
(Csp2 ) or 1.5Ueq (Csp3 )] using a riding model with aromatic C–H bond
length of 0.93 Å, methyl C–H one of 0.96 Å and methylene C–H one
of 0.97 Å. The H atoms linked to nitrogen of the methanesulfonamide
group werelocated by difference Fourier synthesis and wereset as iso-
tropic. Crystal, collection and structure refinement data are summa-
Dcalcd (Mg/m3)
Absorption coefficient (mmꢀ1
F(000)
)
752
Crystal size (mm3)
0.16 ꢂ 0.08 ꢂ 0.03
3.19–27.48
ꢀ12 6 h 6 14,
ꢀ10 6 k 6 10,
ꢀ22 6 l 6 21
24,557
3709 [R(int) = 0.0620]
99.3%
0.9931 and 0.9638
Full-matrix least-squares on F2
3709/0/231
1.016
h Range for data collection (°)
Index ranges
Reflections collected
Independent reflections
Completeness to h = 27.48°
Max. and min. transmission
Refinement method
rized in Table 4. Tables were generated by WINGX32 and the structure
Data/restraints/parameters
Goodness-of-fit on F2
33
representations by ORTEP-316 and MERCURY
.
Final R indices [I > 2
r
(I)]
R1 = 0.0474, wR2 = 0.1072
R1 = 0.0973, wR2 = 0.1308
0.203 and ꢀ0.396
Atomic coordinates, bond lengths, angles and thermal parame-
ters have been deposited at the Cambridge Crystallographic Data
Center, deposition number CCDC 658937.
R indices (all data)
Largest diff. peak and hole (e Åꢀ3
)
lection. One millilitre aliquots were immediately transferred to ep-
pendorff tubes containing of vehicle (DMSO) or test
compounds with concentrations ranging from 0.01 to 500 M.
The tubes were vortexed and incubated in 37 °C under constant
agitation in a water bath for 1 h to induce spontaneous blood clot.
At the end of the incubation the samples was centrifuged at
13,000 rpm for 5 min. Serum samples were collected and stored
in eppendorf tubes at ꢀ20 °C. TXB2 production was determined
using an enzyme immuno-assay kit (Amershan Company) accord-
ing to the manufacturer’s instructions.
4.3. Pharmacology
4 lL
l
4.3.1. Carragenaan-induced rat paw edema and hyperalgesia
assay34,35
Fasted Wistar rats of both sexes (150–200 g) were used. Com-
pounds were administered orally (300 lmol/kg; 0.1 mL/20 g) as a
suspension in 5% arabic gum in saline (vehicle). Control animals re-
ceived an equal volume of vehicle. One hour later, the animals were
injected with either 0.1 mL of 1% carrageenan solution in saline
(1 mg/paw) or sterile saline (NaCl 0.9%), into the subplantar surface
of one of the hind paws respectively. The paws volumes were mea-
sured temporally and at the 3rd hour after the subplantar injection
using a glass plethysmometer coupled to a peristaltic pump. The
edema was calculated as the volume variation between the carra-
4.4.2. In vitro COX-2 assay22
Fresh whole blood was obtained by venipuncture from volun-
teers in syringes containing heparin (10 UI/ml). One millilitre
samples of blood were transferred to eppendorf tubes containing
geenan and saline treated paw. Nimesulide (300 lmol/kg) was used
4
l
L of DMSO (vehicle) or test compounds to obtain concentra-
tions ranging from 0.001 to 1000 M. The tubes were vortexed
and incubated under constant agitation in a water bath for
15 min followed by a new incubation with 20 L of lipopolysac-
charide (LPS) (Sigma–Aldrich, LPS from Escherichia coli serotype
055:B5) at a final concentration of 100 g/ml under constant agi-
as standard drug in the same conditions. Anti-inflammatory activity
was expressed as % of inhibition of the edema when compared with
the vehicle control group. The thermal hyperalgesia is determined
using the modified hot-plate test35 in the same animals used for ede-
ma assay. Rats are placed individually on a hot plate with the tem-
perature adjusted to 51 °C. The latency of the withdrawal response
of the left hind paw is determined at 0, 30, 60, 120, 180 and
240 min post-challenge. The time of maximum permanence permit-
ted on the hot surface is 20 s. Hyperalgesia to heat is defined as a de-
l
l
l
tation during 5 h in order to stimulate COX-2 expression from
blood monocytes. Appropriate controls were done replacing
20 lL of LPS by 20 lL of PBS. At the end of the incubation, the
blood was centrifuged at 13,000 RPM for 5 min to obtain plasma.
The plasma samples were collected and stored at ꢀ20 °C for fur-
ther determination of TXB2 using an enzyme immuno-assay kit
(Amershan Company) according to the manufacturer’s
instructions.
crease in withdrawal latency and calculated as follows:
withdrawal latency (s) = (left paw withdrawal latency at time
0) ꢀ (left paw withdrawal latency at the others times).
D paw
4.4. Human whole blood assay
4.4.3. Formalin-induced pain in mice
4.4.1. In vitro COX-1 assay21
Briefly, fresh whole blood was obtained by venipuncture from
volunteers who had no apparent inflammatory conditions and
had not taken any NSAIDs for at least 2 weeks prior to blood col-
The formalin induced pain test was carried out as described by
Hunskaar and Hole.36 Animals were injected subplantarly with
20
l
L of 2.5% formalin in hind paw. LASSBio 930 (1c) and nimesu-
mol/kg. The time that
lide were administered at a dose of 300
l

J. L. M. Tributino et al. / Bioorg. Med. Chem. 17 (2009) 1125–1131
1131
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References and notes
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