Design, synthesis, antimicrobial activity and molecular modeling studies of novel benzofuroxan derivatives against Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmc.2009.03.011

Molecular modification is a quite promising strategy in the design and development of drug analogs with better bioavailability, higher intrinsic activity and less toxicity. In the search of new leads with potential antimicrobial activity, a new series of 14 4-substituted [N′-(benzofuroxan-5-yl)methylene]benzohydrazides, nifuroxazide derivatives, were synthesized and tested against standard and multidrug-resistant Staphylococcus aureus strains. The selection of the substituent groups was based on physicochemical properties, such as hydrophobicity and electronic effect. These properties were also evaluated through the lipophilic and electrostatic potential maps, respectively, considering the compounds with better biological profile. Twelve compounds exhibited similar bacteriostatic activity against standard and multidrug-resistant strains. The most active compound was the 4-CF₃ substituted derivative, which presented a minimum inhibitory concentration (MIC) value of 14.6-13.1 μg/mL, and a Clog P value of 1.87. The results highlight the benzofuroxan derivatives as potential leads for designing new future antimicrobial drug candidates.

Full text of DOI:10.1016/j.bmc.2009.03.011

Bioorganic & Medicinal Chemistry 17 (2009) 3028–3036
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, antimicrobial activity and molecular modeling studies
of novel benzofuroxan derivatives against Staphylococcus aureus
a
b
Salomão Dória Jorge a, , Andrea Masunari , Carlota Oliveira Rangel-Yagui ,
*
Kerly Fernanda Mesquita Pasqualoto a, Leoberto Costa Tavares a
a Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmacy, University of São Paulo, Av. Prof Lineu Prestes, 580, São Paulo, SP 05508-900, Brazil
b Department of Pharmacy, Faculty of Pharmacy, University of São Paulo, SP 05508-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecular modification is a quite promising strategy in the design and development of drug analogs with
better bioavailability, higher intrinsic activity and less toxicity. In the search of new leads with potential
antimicrobial activity, a new series of 14 4-substituted [N0-(benzofuroxan-5-yl)methylene]benzohydraz-
ides, nifuroxazide derivatives, were synthesized and tested against standard and multidrug-resistant
Staphylococcus aureus strains. The selection of the substituent groups was based on physicochemical
properties, such as hydrophobicity and electronic effect. These properties were also evaluated through
the lipophilic and electrostatic potential maps, respectively, considering the compounds with better bio-
logical profile. Twelve compounds exhibited similar bacteriostatic activity against standard and multi-
drug-resistant strains. The most active compound was the 4-CF3 substituted derivative, which
Received 29 December 2008
Revised 6 March 2009
Accepted 9 March 2009
Available online 13 March 2009
Keywords:
Benzofuroxan derivatives
Molecular modification
MIC
presented a minimum inhibitory concentration (MIC) value of 14.6–13.1 lg/mL, and a ClogP value of
MRSA
1.87. The results highlight the benzofuroxan derivatives as potential leads for designing new future anti-
VISA
Molecular modeling
microbial drug candidates.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The emergence of serious staphylococcal infections with reduced
susceptibility to vancomycin highlights the need for more antimi-
Staphylococcus aureus has been recognized as an important
pathogen of humans. Infections caused by this bacteria species
can lead to serious consequences, especially in hospitalized pa-
tients. The appearance of methicillin resistant strains of S. aur-
eus—MRSA—has been a concern all over the world. This situation
becomes worse and more threatening regarding the ease of trans-
mission of the pathogen among individuals, resulting in the dis-
crobial therapeutic alternatives with increased potency or enhanced
bactericidal activity against MRSA, VISA (vancomycin-intermediate
S. aureus) andVRSA. Only twonewclasseshavebeenintroducedover
the past few decades: the oxazolidinones and the cyclic lipopep-
tides.7 Considering the antimicrobial agents more recently approved
for clinical use, just daptomycin (a cyclic lipopeptide), linezolid (an
oxazolidinone), and tigecycline (a glycylcycline), present activity
against Gram-positive organisms, including MRSA.8
semination of MRSA.1 The resistance to
a great number of
antibiotics presented by MRSA makes it difficult to control in hos-
pital environments.2 In addition to cross-resistance to all beta-lac-
tamic antibiotics, MRSA has also acquired resistance to macrolides,
aminoglycosides, tetracycline, riphampicin and quinolones,
remaining the glycopeptides, such as vancomycin, as some of the
few therapeutical options.3 Nevertheless, the extensive use of gly-
copeptides in the past has led to the emergence of glycopeptide-
resistant organisms and, consequently, there are recommendations
to restrict the use of these agents in the absence of any strong indi-
cations.4,5 The first clinical high-level vancomycin-resistant S. aur-
eus—VRSA—was isolated in 1998.6
In view of the scenario presented above, the search for new and
effective antimicrobial agents, resistant to the mechanisms of de-
fense of these bacteria, is of paramount importance. It is well
known that the discovery of new drugs having completely new
chemical structures is very expensive, involves years of research,
and demands the effort of multi-disciplinary teams.9,10 Therefore,
the optimization of drugs already available can be a good and
financially accessible alternative and could provide better and
promising antimicrobial agents.11
Since the introduction of nitrofurazone, 2-((5-nitro-2-fura-
nyl)methylene)hydrazine carboxamide, in the 40s,12 hundreds of
derivative compounds of 5-nitrofuran were synthesized and evalu-
ated for the antimicrobial activity. These compounds have demon-
strated a large spectrum of action, including Gram-negative and
Gram-positive bacteria and also some protozoa.13 It is important
* Corresponding author. Tel.: +55 11 30913693; fax: +55 11 38156386.
E-mail address: sdjorge@usp.br (S.D. Jorge).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.011

S. D. Jorge et al. / Bioorg. Med. Chem. 17 (2009) 3028–3036
3029
to point out that some of these compounds are not therapeutically
their physicochemical properties such as electronic effect (
hydrophobicity (p
benzoic acids 1a–n, commercially available, were converted into
the respective methyl esters 2a–n by esterification. Ammonolysis
reaction of 2a–n with hydrazine yielded 3a–n. Following, Schiff’s
bases were obtained reacting 3a–n with 5-formylbenzofuroxan, gi-
ven 4a–n.
The 5-formylbenzofuroxan 7 employed in the last step of the
synthesis of the analogues was synthesized by two different routes
in order to evaluate which one would be better (Scheme 2). Route
A, 4-chloro-3-nitrobenzaldehyde 5 was heated with sodium azide
in dimethylsulfoxide to obtain 4-azido-3-nitrobenzaldehyde 6.
Cyclocondensation of this azide in boiling toluene yielded the 5-
formylbenzofuroxan.24 In another effort, route B, the intermediate
was obtained starting from 4-chloro-3-nitrobenzaldehyde in a
one-pot synthesis by nucleophilic substitution of chlorine by azido
group followed by in situ cyclization under solid–liquid phase
transfer catalysis conditions.25 The aldehyde 7 was synthesized
from the corresponding 5 by stirring them at 60 °C with a suspen-
sion of powdered sodium azide in dichloroethane in the presence
of benzyl tributylammonium bromide as a phase-transfer catalyst.
r
) and
employed owing to their undesirable side effects.14,15 Neverthe-
less, more recently, a great interest in nitro-heterocyclic com-
pounds has risen again and new compounds are being studied.16,17
The mechanism of action of nitrofuran compounds is not fully
elucidated. Previous studies suggest that the biological activity of
these compounds is related to the reduction of the nitro group with
formation of free radical toxic species.18 Nifuroxazide, a 5-nitrofu-
ran compound, presents a chemical structure that favors chemical
modifications by means of molecular rational planning. Therefore,
one can envision this drug as a lead molecule for the development
of new analogues. In this work, 14 compounds structurally analo-
gous of nifuroxazide, (Fig. 1A), were designed and evaluated for
antimicrobial activity against S. aureus. The replacement of the
nitrofuran system by benzofuroxan (benzo[1,2-c]1,2,5-oxadiazole
N-oxide) (Fig. 1B) was based on the pharmacophore similarity
attributed to the nitro group, and also the possibility of the N-oxide
moiety acts as a bioreducible group.19–22
), employing the Craig diagram.23 Substituted
A molecular modeling approach was also performed to verify
possible qualitative property–activity relationships, considering
the lipophilicity (LP) and electrostatic potential (EP) maps of the
compounds which presented better biological profile. The lipophil-
icity or hydrophobic property is quite important to the diffusion of
the compounds through the bacterial biological system, and is
associated to the respective calculated logP (ClogP) values. Other-
wise, the EP is related to the electronic density distribution and
considers the contribution of substituent groups to the whole
molecular electronic density. The steric atomic hindrance was also
calculated to verify the substituent volume contribution and its
relationship with the biological data.
2.2. Biological activity evaluation
The minimal inhibitory concentration (MIC) was determined by
the broth microdilution method against S. aureus standard strain
ATCC25923 and multidrug-resistant strains 3SP/R33 and VISA3.26
Table 1 presents the results MIC for the compounds synthesized.
3. Discussion
2. Results
In this study, 14 new compounds were obtained and identified.
All the compounds were synthesized in three steps starting from
the corresponding benzoic acids, such as esterification, ammonol-
ysis, and formation of Schiff’s bases. The esterification step resulted
in satisfactory yields for all the compounds, with values around
90%. However, lower yields, around 50%, were obtained for the
ammonolysis step, owing to the distinct reactivity among methyl
esters under the influence of the substituent groups, as well as to
the difficulties associated with crystallization. The identification
of methyl esters and benzhydrazides was carried out comparing
the experimental melting points to the values previously described
in the literature. Nevertheless, the yields obtained in these first
stages were in agreement with previous results.13,16,27 The classic
method of Schiff’s base formation was employed as the last step.
This reaction is of ease execution, and yields around 85% were ob-
tained. The aldehydes employed in this stage were previously syn-
thesized as described in the literature.24,25
2.1. Synthesis of the nifuroxazide analogues
The designed compounds were obtained as shown in Scheme 1.
The choice of substituent groups was based on the influence of
The compounds 4a–n were isolated as mixture of tautomers at
room temperature (Fig. 2A) that was observed through the corre-
sponding 1H NMR spectra which showed broad signals in the char-
acteristic aromatic zone (Fig. 2B). The proportion of both tautomers
depends on several factors (i.e., solvent, temperature, nature and
position of the substituent of the ring).28–31 The 1H NMR and 13C
NMR spectra performed at higher temperature (Fig. 2C and D)
makes it possible to determine which one of isomers predominate.
The coupling constants and chemical shifts showed the presence of
one isomer only, which is the 1,5-disubstituted heterocycle.
In order to evaluate the activity against S. aureus, the MIC of the
compounds obtained were determined employing the successive
microdilution method.26 Owing to the low solubility of the com-
pounds in the culture media, it was necessary to use DMSO as a
solvent, but not exceeding 5% of the total. Concentrations of DMSO
ranged from 5.0% to 0.01% in phase I and ranged from 2.5% to 1.0%
in phase II. All derivatives presented MIC values with concentra-
Figure 1. Chemical structures of nifuroxazide (A), pointing out the groups where
chemical modifications were carried out in the present study, and of benzofuroxan
derivatives (B).

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S. D. Jorge et al. / Bioorg. Med. Chem. 17 (2009) 3028–3036
Scheme 1. Synthesis of benzofuroxan derivatives.
MIC = 29.1–26.2 lg/mL. The compounds 4-N(CH3)2 (4j) and 4-
SO2NH2 (4m) substituted derivatives did not show any antimicro-
bial activity at the concentrations studied and it was not possible
to increase the initial stock solution concentration of these com-
pounds without increasing the concentration of dimethylsulfoxide
used in the test.
The influence of the substituent group’s physicochemical prop-
erties on the activity of the compounds was observed. More specif-
ically, hydrophobicity was found to be directly related to the
antimicrobial activity, in agreement with other studies carried
out for a different series of nifuroxazide analogs.13,16,27 This prop-
erty is related to the ability of a compound to diffuse through the
biological membranes and reach its site of action. The presence
of hydrophobic substituents attached to a benzene moiety, such
as 4-Cl (4h), 4-CF3 (4k), and 4-Br (4l), provided a positive influence
on antimicrobial activity. The electronic effect also played a role in
activity, as can be seen for the compounds having an electron do-
nor character, such as 4-NH2 (4c), and 4-OCH3 (4g).
Scheme 2. Synthesis of 5-formylbenzofuroxan by two different routes.
One interesting observation is that the two most active com-
pounds, 4-CF3 4k (MIC = 14.6–13.1
lg/mL) and 4-NH2 4c
tions of DMSO much lesser than those needed to kill the microor-
ganism (MIC = 12.5–11.0%, Table 1). Thus, the completely inhibi-
tion of S. aureus growth refers only to the intrinsic bacteriostatic
activity of compounds, and any possibility of synergistic effect be-
tween the compounds and DMSO was discharged.
(MIC = 17.0–15.3 g/mL), possess completely distinct properties.
l
While the –CF3 substituent group presents a hydrophobic charac-
ter and an electron withdrawing effect, the –NH2 substituent group
has a more hydrophilic character and an electron donor effect. The
possible explanation to this fact would be based on the volume
similarity of these two substituent groups. If the steric effect plays
an important role in this portion of molecule, perhaps this could
partially explain why the –NH2 presents good activity despite it
is hydrophilic character.
A molecular modeling approach was carried out to investigate
the lipophilic, electronic, and steric hindrance properties of the
two most active compounds, 4k and 4c. The LP and EP maps were
calculated onto the molecular surfaces and are presented in Figures
3 and 4, respectively. The 4k LP map shows a bigger brown region
(hydrophobic), which is corroborated by its ClogP value (1.87) (see
Fig. 3).
The EP maps are in agreement to the electronic effect of the –
CF3 and NH2 substituent groups, already mentioned. The –CF3
(4k) region presents higher electronic density (green/yellow color)
than the –NH2 (4c) (blue color) (see Fig. 4).
Considering the assumption that relates the better activity of 4k
and 4c to the similar volume of their substituent groups, the atom-
ic steric hindrance index was calculated. The visualization of the
substituent group volumes are presented in Figure 5. The total ste-
All compounds were tested against the three strains,
ATCC25923, SP3/R33 and VISA3. The SP3/R33 and VISA3 strains
of S. aureus are resistant to 19 antimicrobial agents currently avail-
able in the market, differing only in susceptibility to vancomy-
cin.32,33 The SP3/R33 is susceptible to vancomycin (MIC ꢀ 2
lg/
mL), while the VISA3 strain presents intermediate resistance to
vancomycin (MIC ꢁ 8
dards Institute defines staphylococci requiring vancomycin con-
centrations lower than g/mL for growth inhibition as
susceptible, those requiring 8–16 g/mL as intermediate resis-
tance, and those requiring concentrations over 32 g/mL as resis-
lg/mL). The Clinical and Laboratory Stan-
4
l
l
l
tant.26 These two resistant strains used in our study were
isolated from patients in hospitals of the city of São Paulo and were
characterized by Pulsed Field Gel Electrophoresis (PFGE), showing
the same PFGE profile as the Brazilian Endemic Clone (BEC).32,33
The MIC results obtained are presented in Table 1. Accordingly,
the most active compound was the 4-CF3 (4k) substituted deriva-
tive with MIC = 14.6–13.1
lg/mL, while the lowest activity was ob-
served for 4-OH (4d) and 4-CN (4f) substituted derivatives with

S. D. Jorge et al. / Bioorg. Med. Chem. 17 (2009) 3028–3036
3031
Table 1
Minimal inhibitory concentration values of benzofuroxan derivatives and drug controls against Staphylococcus aureus standard strain ATCC25923 and multidrug-resistant strains
3SP/R33 and VISA3
Compound
R
ATCC25923
MIC (
g/mL)c
3SP/R33a
VISA3a,b
l
MIC (
l
g/mL)c
MIC (l
g/mL)c
Phase I
Phase II
Phase I
Phase II
Phase I
Phase II
4a
4b
4c
4d
4e
4f
H
20.0–10.0
40.0–20.0
20.0–10.0
40.0–20.0
20.0–10.0
40.0–20.0
20.0–10.0
20.0–10.0
40.0–20.0
>80.0
18.0–16.2
23.3–21.0
17.0–15.3
29.1–26.2
16.2–14.6
29.1–26.2
18.0–16.2
17.0–15.3
23.6–21.2
—
20.0–10.0
40.0–20.0
20.0–10.0
40.0–20.0
20.0–10.0
40.0–20.0
20.0–10.0
20.0–10.0
40.0–20.0
>80.0
18.0–16.2
23.3–21.0
17.0–15.3
29.1–26.2
16.2–14.6
29.1–26.2
18.0–16.2
17.0–15.3
23.6–21.2
—
20.0–10.0
40.0–20.0
20.0–10.0
40.0–20.0
20.0–10.0
40.0–20.0
20.0–10.0
20.0–10.0
40.0–20.0
>80.0
18.0–16.2
23.3–21.0
17.0–15.3
29.1–26.2
16.2–14.6
29.1–26.2
18.0–16.2
17.0–15.3
23.6–21.2
—
CH3
NH2
OH
F
CN
OCH3
Cl
COCH3
N(CH3)2
CF3
BR
SO2NH2
I
4g
4h
4i
4jd
4k
4l
20.0–10.0
20.0–10.0
>80.0
14.6–13.1
20.0–18.0
—
20.0–10.0
20.0–10.0
>80.0
14.6–13.1
20.0–18.0
—
20.0–10.0
20.0–10.0
>80.0
14.6–13.1
20.0–18.0
—
4md
4n
40.0–20.0
26.2–23.6
40.0–20.0
26.2–23.6
40.0–20.0
26.2–23.6
Ampicilin
0.2–0.1
4.0–2.0
1.0–0.5
32.0–16.0
64.0–32.0
1.0–0.5
32.0–16.0
64.0–32.0
8.0–4.0
Chloramphenicol
Vancomycin
DMSO (%)
12.5–11.0
12.5–11.0
12.5–11.0
a
Resistant to amoxicillin/clavulanic acid, ampicillin, cephazoline, cephotaxime, cephalotine, ciprofloxacin, clindamycin, erythromycin, gentamicin, imipenem, nitrofu-
rantoin, norfloxacin, oxacillin, penicillin, rifampicin, trimethoprim/sulfametoxazole.
b
Vancomycin-intermediate Staphylococcus aureus strain.
Values corresponding to the average of 3 repetitions.
c
ric hindrance index found for –CF3 and –NH2 groups are 2.97 and
2.59, respectively, suggesting indeed some proximity of their
volumes.
In order to better understand the influence of physicochemical
properties on the activity of this new class of compounds, we are
currently working on the synthesis of new analogues. The exten-
sion of the series will allow us, in a near future, to establish quan-
titative structure activity–relationships for this class and,
therefore, help on the search of new analogues.
The MIC values against the multidrug-resistant strains were
similar to those observed against the ATCC standard strain. There-
fore, we can suggest that the action mode of these compounds
against the three strains tested is quite the same, and the multi-
drug-resistant strains did not develop any mechanisms of resis-
tance to the benzofuroxan derivatives synthesized in this work.
Although the MIC values of the compounds against the ATCC strain
were higher than the antibiotics used as control, the benzofuroxan
derivatives showed remarkable activity against both multidrug
resistant strains, with MIC values higher than ampicilin and clo-
ramphenicol, but lower than vancomycin. These findings reinforce
the potential of these compounds as promising leads for the devel-
opment of new drugs against multidrug-resistant S. aureus, as well
as other pathogens.
pounds. The benzofuroxan derivatives presented activity against
standard and multidrug-resistant strains of S. aureus, except 4-
N(CH3)2 (4j) and 4-SO2NH2 (4m). The MIC results indicated that
the antimicrobial activity of the investigated compounds is
influenced by the physicochemical properties of the substituent
group at position 4 in the benzene ring. More specifically, there
seems to be a direct relationship between activity and hydro-
phobic property. The results highlight these novel benzofuroxan
derivatives as potential leads for designing new antimicrobial
drug candidates.
5. Experimental
5.1. Chemistry
IR spectra were recorded on a Shimadzu IR—470 spectrometer,
using KBr pellets. NMR spectra were recorded on a BRUKER ADPX
Advanced (300 MHz) spectrometer employing DMSO-d6 solutions
with tetramethylsilane as internal standard. Melting points were
determined using Micro-Química MQAPF-301 apparatus and ele-
mental analysis was performed on a Perkin–Elmer 24013 CHN Ele-
mental Analyzer.
5.1.1. General procedure for the preparation of methyl esters
(2a–n)
4. Conclusions
Each substituted benzoic acid 1a–n (0.04 mol) was refluxed for
4 h in 50.0 mL (1.23 mol) of anhydrous methanol and 1.0 mL
(2.0 mmol) of sulfuric acid. The solvent was evaporated and the
product obtained washed with cold water.
In this study, 14 novel benzofuroxan derivatives were syn-
thesized, identified and biological assayed. All synthesis reac-
tions presented satisfactory yields and the melting point
determinations attested the high degree of purity of the com-

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S. D. Jorge et al. / Bioorg. Med. Chem. 17 (2009) 3028–3036
Figure 2. (A) Tautomeric equilibrium of benzofuroxan derivatives. (B) 1H NMR spectra of 4a in DMSO-d6 at 298 K. (C) 1H NMR spectra of 4a in DMSO-d6 at 348 K. (D) HETCOR
spectra of 4a in DMSO-d6 at 348 K.
Figure 3. LP translucid and opaque colored maps of the benzofuroxan derivatives 4c and 4k, respectively, using SYBYL 8.0 (Tripos, Inc., 2007).42 Brown color indicates
hydrophobic regions and blue color denotes hydrophilic areas. The molecules are displayed in stick capped model (carbon atoms are in light gray color, oxygen in red,
nitrogen in blue, fluorine in green, and hydrogen atoms are presented in cyan).

S. D. Jorge et al. / Bioorg. Med. Chem. 17 (2009) 3028–3036
3033
Figure 4. EP translucid and opaque zipped colored maps of the benzofuroxan derivatives 4c and 4k, respectively, using GAUSSVIEW 3.0 (Gaussian Inc. 1995–2003). Red color
indicates negative values of EP (higher electronic density) while blue color denotes positive values (lower electronic density) in a color range from ꢂ7.54 to 7.54 eꢂ2. The
molecules are displayed in stick capped model (carbon atoms are in gray color, oxygen in red, nitrogen in blue, fluorine in light blue, and hydrogen atoms are presented in
white).
Figure 5. Visualization of the substituent groups’ volumes for the benzofuroxan derivatives 4c and 4k, respectively (ViewerLite 5.0, Accelrys Inc.: 2002). The molecules are
displayed in stick capped model (carbon atoms are in gray color, oxygen in red, nitrogen in blue, fluorine in green, and hydrogen atoms are presented in white). The
substituent groups are showed as CPK models and van der Waals surfaces.
5.1.2. Generalprocedure for thepreparation of benzohydrazides
(3a–n)
cooled, poured into 100.0 mL of water, and extracted with ether.
After drying with MgSO4 and evaporating the solvent an yellow
oil, which solidified at 0 °C, was obtained. It was crystallized from
ethanol to give 4-azido-3-nitrobenzaldehyde 6. Pale yellow plates
Hydrazine hydrate 64% (v/v) (30.0 mL, 0.33 mol) was heated up
to 50–60 °C. The methyl ester 2a–n (0.01 mol) was added and the
mixture was refluxed for 10 min. The cooling was proceeded
sequentially in a water bath, followed by ice bath and dry ice–eth-
anol bath. The solid was filtered and washed with cold water.
(75%); 74.0–75.0 °C. The azide
6 (5 mmol) was refluxed for
30 min in 15 mL of toluene. The solvent was evaporated and the
remaining oil was redissolved in 5.0 mL of boiling EtOAc. Petro-
leum ether (bp 60–80 °C) (20.0 mL) was added and the solution
was cooled to give 5-formylbenzofuroxan 7 (80%). Route B—4-
5.1.3. 5-Formylbenzofuroxan
Route A—a mixture of 4-chloro-3-nitrobenzaldehyde
5
chloro-3-nitrobenzaldehyde 5 (0.01 mol) and sodium azide
(0.020 mol) and sodium azide (0.015 mol) in 30.0 mL of dimethyl-
sulfoxide was heated at 75 °C during 30 min. The solution was
(0.01 mol) were added to a vigorously stirred solution of benzyltri-
butylammonium bromide (1 mmol) in 50.0 mL of 1,2-dichloroeth-

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S. D. Jorge et al. / Bioorg. Med. Chem. 17 (2009) 3028–3036
ane at 25 °C. The mixture was slowly heated up to 60 °C and main-
tained at this temperature for 6 h. The reaction mixture was cooled
to 28 °C and filtered. The filtrate was washed first with 100.0 mL of
1 N hydrochloric acid and subsequently with 50.0 mL of water. The
organic layer was separated and dried with MgSO4. Thesolvent was
distilled and the residue was crystallized from ethanol to yield the
aldehyde 7 (85%). Pale yellow needle crystal; mp 65.0–66.0 °C; IR
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.97 (s, 1H), 8.49 (s, 1H),
8.00 (d, 2H, J = 7.0 Hz), 7.92 (d, 1H, J = 9.4 Hz), 7.84 (s, 1H), 7.71
(d,1H, J = 9.4 Hz), 7.34 (d, 2H, J = 7.9 Hz); 13C NMR {H} (DMSO-d6,
300 MHz): d (ppm):166.4 (C15), 163.1 (C11), 145.4 (C8), 136.6
(C5), 131.05 (C13,C17), 130.9 (C12), 130.1 (C7a), 129.8 (C3a),
125.6 (C6), 115.8 (C14,C16), 113.9 (C7), 112.3 (C4); Anal. Calcd
for (C14H9FN4O3): C, 56.00; H, 3.02; N, 18.66. Found: C, 55.73; H,
3.15; N, 18.19.
m
max: 1691, 1607, 1573, 1520; 1H NMR(DMSO-d6, 300 MHz): d
(ppm): 10.09 (s, 1H), 8.43 (s, 1H), 7.85 (d, 1H, J = 9.4 Hz), 7.78 (d, 1H,
J = 9.4 Hz); 13C NMR {H} (DMSO-d6, 300 MHz): d (ppm): 191.1,
153.2, 136.8, 129.0, 122.1, 119.2, 114.8; Anal. Calcd for (C7H4N2O3):
C, 51.27; H, 2.46; N, 17.07. Found: C, 51.57; H, 2.44; N, 16.93.
5.1.4.6. 4-Cyano-[N0-(benzofuroxan-5-yl)methylene]benzohyd-
razide (4f). Dark yellow solid (92%); mp 204.0–205.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 12.14 (s, 1H), 8.48 (s, 1H),
8.04 (d, 2H, J = 7.6 Hz), 7.96 (d, 2H, J = 8.2 Hz), 7.83 (d, 1H,
J = 9.4 Hz), 7.79 (s, 1H), 7.70 (d, 1H, J = 9.4 Hz); 13C NMR {H}
(DMSO-d6, 300 MHz): d (ppm): 159.8 (C11), 145.4 (C8), 136.8
(C12), 132.8 (C5), 129.6 (C14,C16), 129.3 (C7a), 129.0 (C3a),
128.6 (C13,C17), 127.7 (C6), 118.1 (CN), 117.1 (C15), 116.7 (C7),
115.2 (C4); Anal. Calcd for (C15H9N5O3): C, 58.63; H, 2.95; N,
22.79. Found: C, 58.59; H, 3.01; N, 22.62.
5.1.4. General procedure for the preparation of benzofuroxans
derivatives (4a–n)
A mixture of 5-formylbenzofuroxan 7 (0.01 mol) and benzohyd-
razides 3a–n (0.01 mol) in water, sulfuric acid, acetic acid and
methanol (8:7:8:20 v/v) was heated under reflux for 1 h. After
cooling, the mixture was poured into cold water to give 4a–n.
5.1.4.7. 4-Methoxy-[N0-(benzofuroxan-5-yl)methylene]benzohy
drazide (4g). Yellow solid (93%); mp 211.0–212.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.81 (s, 1H), 8.48 (s, 1H),
7.92 (d, 2H, J = 8.7 Hz), 7.90 (d, 1H, J = 9.4 Hz), 7.79 (s, 1H), 7.70
(d, 1H, J = 9.4 Hz), 7.04 (d, 2H, J = 8.7 Hz), 3.84 (s, 3H); 13C NMR
{H} (DMSO-d6, 300 MHz): d (ppm): 164.0 (C15), 162.7 (C11),
144.5 (C8), 138.2 (C5), 130.7 (C7a), 130.3 (C13,C17), 130.1 (C3a),
129.5 (C6), 125.8 (C12), 116.5 (C7), 114.7 (C4), 114.1 (C14,C16),
55.9 (CH3); Anal. Calcd for (C15H12N4O4): C, 57.69; H, 3.87; N,
17.94. Found: C, 57.19; H, 3.88; N, 17.31.
5.1.4.1. N0-(Benzofuroxan-5-yl)methylene benzohydrazide
(4a). Yellow solid (94%); mp 214.0–215.0 °C. 1H NMR(DMSO-d6,
300 MHz,): d (ppm): 11.97 (s, 1H), 8.53 (s, 1H), 7.95 (d, 1H,
J = 9.5 Hz), 7.94 (d, 2H, J = 6.9 Hz), 7.85 (s, 1H), 7.74 (d, 1H,
J = 9.5 Hz), 7.58 (m, 3H); 13C NMR {H} (DMSO-d6, 300 MHz): d
(ppm):163.8 (C11), 144.2 (C8), 137.3 (C5), 132.9 (C12), 131,7 (C7a),
131,3 (C15), 131.0 (C3a), 128.6 (C6), 127.9 (C14,C16), 127.5
(C13,C17), 115.7 (C7), 114.2 (C4); Anal. Calcd for (C14H10N4O3): C,
59.57; H, 3.57; N, 19.85. Found: C, 59.51; H, 3.58; N, 19.90.
5.1.4.2. 4-Methyl-[N0-(benzofuroxan-5-yl)methylene]benzohy-
drazide (4b). Yellow solid (93%); mp 217.0–218.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.87 (s, 1H), 8.51 (s, 1H),
7.93 (d, 1H, J = 9.5 Hz), 7.83 (d, 2H, J = 8.1 Hz), 7.80 (s, 1H), 7.71
(d, 1H, J = 9.5 Hz), 7.33 (d, 2H, J = 8.0 Hz), 2.39 (s, 3H); 13C NMR
{H} (DMSO-d6, 300 MHz): d (ppm):164.4 (C11), 144.8 (C8), 142.3
(C15), 138.2 (C5), 131.2 (C12); 130.8 (C7a), 129.5 (C3a), 129.3
(C14,C16), 129.0 (C6), 128.4 (C13,C17), 116.5 (C7), 114.9 (C4),
21.3 (CH3); Anal. Calcd for (C15H12N4O3): C, 60.81; H, 4.08; N,
18.91. Found: C, 61.01; H, 4.10; N, 18.93.
5.1.4.8. 4-Chloro-[N0-(benzofuroxan-5-yl)methylene]benzohyd-
razide (4h). Yellow solid (91%); mp 212.0–213.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.97 (s, 1H), 8.46 (s, 1H),
7.90 (d, 2H, J = 8.6 Hz), 7.89 (d, 1H, J = 9.4 Hz), 7.81 (s, 1H), 7.68 (d,
1H, J = 9.4 Hz), 7.55 (d, 2H, J = 8.6 Hz); 13C NMR {H} (DMSO-d6,
300 MHz): d (ppm): 159.8 (C11), 145.3 (C8), 137.9 (C15), 137.2
(C5), 132.4 (C12), 130.3 (C13,C17), 130.2 (C7a), 129.4 (C3a), 128.8
(C14,C16), 127.6 (C6), 116.6 (C7), 115.3 (C4); Anal. Calcd for
(C14H9ClN4O3): C, 53.09; H, 2.86; N, 17.69. Found: C, 53.20; H,
2.83; N, 17.76.
5.1.4.3. 4-Amino-[N0-(benzofuroxan-5-yl)methylene]benzohyd-
razide (4c). Yellow solid (90%); mp 226.0–227.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.59 (s, 1H), 8.48 (s, 1H),
7.96 (d, 1H, J = 9.5 Hz), 7.76 (s, 1H), 7.71 (d, 1H, J = 9.5 Hz), 7.70
(d, 2H, J = 8.5 Hz), 6.64 (d, 2H, J = 8.6 Hz), 5.61 (s, 2H); 13C NMR
{H} (DMSO-d6, 300 MHz): d (ppm): 163.5 (C11), 158.9 (C15),
152.0 (C8), 142.6 (C5), 137.7 (C6), 129.3 (C13,C17), 128.8 (C7a),
128.4 (C3a), 119.2 (C12), 115.5 (C7), 113.3 (C4), 112.5 (C14,C16);
Anal. Calcd for (C14H11N5O3): C, 56.56; H, 3.73; N, 23.56. Found:
C, 56.51; H, 3.70; N, 23.43.
5.1.4.9. 4-Acetyl-[N0-(benzofuroxan-5-yl)methylene]benzohyd-
razide (4i). Yellow solid (87%); mp 229.0–230.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 12.06 (s, 1H), 8.51 (s, 1H),
8.06 (d, 2H, J = 7.9 Hz), 8.01 (d, 2H, J = 7.4 Hz), 7.84 (s, 1H), 7.77
(d, 1H, J = 9.5 Hz), 7.71 (d, 1H, J = 9.5 Hz), 2.62 (s, 3H); 13C NMR
{H} (DMSO-d6, 300 MHz): d (ppm): 197.6 (C@O), 162,5 (C11),
145.5 (C8), 139.1 (C15), 136.6 (C12), 132.0 (C5), 131.1 (C7a),
129.9 (C3a), 128.2 (C14,C15), 128.1 (C13,C17), 126.0 (C6), 117.3
(C7), 114.1 (C4), 26.9 (CH3); Anal. Calcd for (C16H12N4O4): C,
59.26; H, 3.73; N, 17.28. Found: C, 59.42; H, 3.74; N, 17.36.
5.1.4.4. 4-Hydroxy-[N0-(benzofuroxan-5-yl)methylene]ben-
zohydrazide (4d). Yellow solid (84%); mp 295.0–296.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.73 (s, 1H), 10.42 (s,
1H), 8.50 (s, 1H), 7.95 (d, 1H, J = 9.5 Hz), 7.84 (d, 2H,
J = 8.4 Hz), 7.80 (s, 1H), 7.72 (d, 1H, J = 9.5 Hz), 6.89 (d, 2H,
J = 8.4 Hz); 13C NMR {H} (DMSO-d6, 300 MHz): d (ppm):163.5
(C11), 161.4 (C15), 144.4 (C8), 136.9 (C5), 131.1 (C6), 130.4
(C13,C17), 129.9 (C7a), 127.6 (C3a), 123.8 (C12), 118.3 (C7),
115.5 (C14,C16), 113.4 (C4); Anal. Calcd for (C14H10N4O4): C,
56.38; H, 3.38; N, 18.78. Found: C, 55.83; H, 3.44; N, 18.31.
5.1.4.10. 4-Dimethylamino-[N0-(benzofuroxan-5-yl)methylene]
benzohydrazide (4j). Orange solid (82%); mp 214.0–215.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.64 (s, 1H), 8.48 (s, 1H),
7.93 (d, 1H, J = 9.6 Hz), 7.82 (d, 2H, J = 9.0 Hz), 7.74 (s, 1H), 7.69
(d, 1H, J = 9.6 Hz), 6.75 (d, 2H, J = 9.0 Hz), 2.99 (s, 6H); 13C NMR
{H} (DMSO-d6, 300 MHz): d (ppm): 164.1 (C11), 153.2 (C15),
143.6 (C8), 138.5 (C5), 129.9 (C13,C17), 129.6 (C7a), 127.9 (C3a),
126.4 (C6), 119.8 (C12), 116.3 (C7), 114.3 (C4), 111.3 (C14,C16),
41.2 (N(CH3)2); Anal. Calcd for (C16H15N5O3): C, 59.07; H, 4.65; N,
21.53. Found: C, 59.25; H, 4.63; N, 21.32.
5.1.4.5. 4-Fluoro-[N0-(benzofuroxan-5-yl)methylene]benzohyd-
razide (4e). Orange solid (82%); mp 196.0–197.0 °C. 1H
5.1.4.11. 4-Trifluoromethyl-[N0-(benzofuroxan-5-yl)methylene]
benzohydrazide (4k). Yellow solid (82%); mp 201.0–202.0 °C. 1H

S. D. Jorge et al. / Bioorg. Med. Chem. 17 (2009) 3028–3036
3035
NMR(DMSO-d6, 300 MHz,): d (ppm): 12.13 (s, 1H), 8.49 (s, 1H),
8.15 (d, 1H, J = 9.6 Hz), 8.09 (d, 2H, J = 7.7 Hz), 7.87 (d, 2H,
J = 8.9 Hz), 7.86 (s, 1H), 7.71 (d, 1H, J = 9.6 Hz); 13C NMR {H}
(DMSO-d6, 300 MHz): d (ppm): 159.8 (C11), 145.9 (C8), 137.8
(C12), 137.5 (C15), 135.4 (C5), 132.6 (C7a), 131.9 (C3a), 129.2
(C13,C17), 126.1 (C6), 125.7 (C14,C16), 122.5 (CF3), 116.7 (C7),
115.5 (C4); Anal. Calcd for (C15H9F3N4O3): C, 51.44; H, 2.59; N,
16.00. Found: C, 50.95; H, 2.44; N, 15.99.
column 12. Bacterial suspensions were prepared by the same pro-
cedure described in phase I and 0.1 mL of inoculum was trans-
ferred to each well, except for the column 11. The plates were
mixed and incubated at 35 °C for 18 h.
5.3. Molecular modeling
The three-dimensional structures the benzofuroxan derivatives
or ligands, which presented better biological profile (4c and 4k), in
their neutral forms were constructed using the HYPERCHEM 7 soft-
ware.34 The Cartesian coordinates of NX crystallized structure were
retrieved from Cambridge Structural Database (CSD)35 (entry code
LEQTAC (R-factor 0.11)36 and used as a geometry reference in the
building up of the ligands. Each structure was energy-minimized
using the following methods: MM+ force field (derived from
MM2),37 AM1 semiempirical method38 in HYPERCHEM 7,34 and Har-
tree–Fock (basis set 6–31G**) ab initio method in GAUSSIAN 03W,
v.6,39 without any restriction. Electrostatic partial atomic charges
(Chelpg) were computed employing HF/6-31G** method, also
implemented in the GAUSSIAN 03W program.
5.1.4.12. 4-Bromo-[N0-(benzofuroxan-5-yl)methylene]benzohy-
drazide (4l). Yellow solid (90%); mp 210.0–211.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 11.97 (s, 1H), 8.46 (s, 1H),
7.88 (d, 1H, J = 9.1 Hz), 7.83 (d, 2H, J = 8.5 Hz), 7.81 (s, 1H), 7.70
(d, 2H, J = 8.2 Hz), 7.68 (d, 1H, J = 9.1 Hz); 13C NMR {H} (DMSO-d6,
300 MHz): d (ppm): 159.8 (C11), 145.4 (C8), 138.0 (C5), 132.9
(C12), 131.8 (C14,C16), 130.6 (C7a), 130.4 (C13,C17), 129.9 (C3a),
129.4 (C6), 126.0 (C15), 116.4 (C7), 115.4 (C4); Anal. Calcd for
(C14H9BrN4O3): C, 46.56; H, 2.51; N, 15.51. Found: C, 46.61; H,
2.63; N, 15.56.
5.1.4.13. 4-Sulfamoyl-[N0-(benzofuroxan-5-yl)methylene]ben-
zohydrazide (4m). Yellow solid (97%); mp 272.0–273.0 °C. 1H
NMR(DMSO-d6, 300 MHz,): d (ppm): 12.00 (s, 1H), 8.46 (s, 1H),
8.03 (d, 2H, J = 8.1 Hz), 7.94 (d, 2H, J = 8.1 Hz), 7.79 (s, 1H), 7.77
(d, 1H, J = 9.3 Hz), 7.65 (d, 1H, J = 9.3 Hz), 7.28 (s, 2H); 13C NMR
{H} (DMSO-d6, 300 MHz): d (ppm): 164.2 (C11), 148.5 (C15),
147.4 (C8), 136.7 (C12), 135.5 (C5), 132.6 (C7a), 131.9 (C3a),
129.0 (C13,C17), 126.1 (C14,C16), 123.9 (C6), 116.7 (C7), 115.4
(C4); Anal. Calcd for (C14H11N5O5S): C, 46.54; H, 3.07; N, 19.38.
Found: C, 46.45; H, 3.05; N, 19.62.
The structures modeled as described above were taken as the
initial structures for computing the LP property onto a Connolly
molecular surface,40,41 using a sphere probe of 1.4 Å radii (SYBYL
8.0 package).42 The LP property and the ClogP values were calcu-
lated employing the Ghose and co-workers method43 and Sybyl
Line Notation (SLN) (SYBYL 8.0 package).42 Properties that are ap-
plied to a surface can be a useful analytic tool in visually identify-
ing areas of interest on the surface. The resulting LP maps were
analyzed according to the color ramps, which range from brown
(highest lipophilic area of the molecule) to blue (highest hydro-
philic area). The color scheme is easy to interpret if the blue is asso-
ciated with water and the brown as oil/fat.
The EP property was computed in a cube surface using an iso-
value of 0.0004 employing the GAUSSIAN 03W program.39 Negative
values of EP (higher electronic density) are denoted in red while
positive values are mapped in blue (lower electronic density) on
the molecular surfaces.
5.1.4.14. 4-Iodo-[N0-(benzofuroxan-5-yl)methylene]benzohyd-
razide (4n). Yellow solid (87%); mp 228.0–229.0 °C. 1H NMR
(DMSO-d6, 300 MHz,): d (ppm): 11.98 (s, 1H), 8.47 (s, 1H), 7.90
(d, 2H, J = 8.4 Hz), 7.88 (d, 1H, J = 9.7 Hz), 7.82 (s, 1H), 7.70 (d,
1H, J = 9.7 Hz), 7.68 (d, 2H, J = 8.2 Hz); 13C NMR {H} (DMSO-d6,
300 MHz): d (ppm): 159.8 (C11), 145.4 (C8), 138.0 (C12), 137.8
(C14,C16), 133.2 (C5), 131.8 (C7a), 130.3 (C13,C17), 130.0 (C3a),
129.4 (C6), 116.7 (C7), 115.3 (C4), 99.5 (C15); Anal. Calcd for
(C14H9IN4O3): C, 41.20; H, 2.22; N, 13.73. Found: C, 41.68; H,
2.36; N, 13.71.
The atomic steric hindrance of the ligands 4c and 4k were cal-
culated from the covalent radii values and geometrical distances,
using the MARVIN VIEW program.44 This property is additive, which
means the total steric hindrance of a substituent group corre-
sponds to the sum of each atomic steric hindrance calculated.44
5.2. Biology
Acknowledgements
Phase I—Minimal Inhibitory Concentration (MIC) of the com-
pounds was determined with 96-well microtiter plates containing
twofold serial dilutions of the compounds in Tryptic Soy Broth
(TSB-SigmaÒ) medium.26 Stock solutions of the compounds were
prepared in DMSO/TSB 1:10 v/v. Concentrations ranged from 0.1
The authors thank Professor Elza Masae Mamizuka, from the
Department of Clinical and Toxicological Analysis-USP, for provid-
ing the 3SP/R33 and VISA3 S. aureus strains; and, the Brazilian Gov-
ernmental Agencies CNPq and CAPES for the financial support.
to 80 lg/mL, using ampicilin, chloramphenicol and vancomycin
as drug controls. Bacterial suspensions were prepared by turbidity
adjustment to a density of 0.5 on the McFarland scale and further
dilution in sterile physiologic saline solution and TSB. The plates
were incubated at 35 °C for 18 h, and the lowest concentration of
compound at which there was no visible growth was considered
the MIC. Readings at 24 and 48 h were carried out for sterility con-
trol. Experiments were performed in triplicate.
Phase II—this phase aimed at narrowing the MIC values that
were defined in phase I. Stock solutions (1 mL) were prepared
using twofold the MIC value determined in phase I. A volume of
0.1 mL of this solution was added to the column 1 of microplate.
Then 0.1 mL of TSB was added to the initial stock solution diluting
it up to 10%. After mixed, 0.1 mL of this new solution was added to
column 2. Then 0.1 mL of TSB was added to the solution diluting it
once more up to 10%. This procedure was repeated till the 11th col-
umn. For the positive growth control, 0.1 mL of TSB was added to
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