Novel benzofuroxan derivatives against multidrug-resistant Staphylococcus aureus strains: Design using Topliss' decision tree, synthesis and biological assay

Article abstract of DOI:10.1016/j.bmc.2011.06.034

The aim of this study was the design of a set of benzofuroxan derivatives as antimicrobial agents exploring the physicochemical properties of the related substituents. Topliss' decision tree approach was applied to select the substituent groups. Hierarchical cluster analysis was also performed to emphasize natural clusters and patterns. The compounds were obtained using two synthetic approaches for reducing the synthetic steps as well as improving the yield. The minimal inhibitory concentration method was employed to evaluate the activity against multidrug-resistant Staphylococcus aureus strains. The most active compound was 4-nitro-3-(trifluoromethyl)[N′-(benzofuroxan-5-yl) methylene]benzhydrazide (MIC range 12.7-11.4 μg/mL), pointing out that the antimicrobial activity was indeed influenced by the hydrophobic and electron-withdrawing property of the substituent groups 3-CF₃ and 4-NO₂, respectively.

Full text of DOI:10.1016/j.bmc.2011.06.034

Bioorganic & Medicinal Chemistry 19 (2011) 5031–5038
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel benzofuroxan derivatives against multidrug-resistant Staphylococcus
aureus strains: Design using Topliss’ decision tree, synthesis and biological assay
Salomão Dória Jorge a, , Fanny Palace-Berl a, Andrea Masunari b, Cléber André Cechinel c, Marina Ishii a,
⇑
Kerly Fernanda Mesquita Pasqualoto a, Leoberto Costa Tavares a
a Departamento de Tecnologia Bioquímico Farmacêutica, Faculdade de Farmácia, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil
b Centro Universitário São Camilo, Faculdade de Farmácia, São Paulo, SP 04263-200, Brazil
c Faculdade de Tecnologia e Ciências, Diretoria de Pesquisa e Pós-Graduação, Salvador, BA 41714-590, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this study was the design of a set of benzofuroxan derivatives as antimicrobial agents explor-
ing the physicochemical properties of the related substituents. Topliss’ decision tree approach was
applied to select the substituent groups. Hierarchical cluster analysis was also performed to emphasize
natural clusters and patterns. The compounds were obtained using two synthetic approaches for reduc-
ing the synthetic steps as well as improving the yield. The minimal inhibitory concentration method was
employed to evaluate the activity against multidrug-resistant Staphylococcus aureus strains. The most
active compound was 4-nitro-3-(trifluoromethyl)[N0-(benzofuroxan-5-yl)methylene]benzhydrazide
(MIC range 12.7–11.4 lg/mL), pointing out that the antimicrobial activity was indeed influenced by
the hydrophobic and electron-withdrawing property of the substituent groups 3-CF3 and 4-NO2,
respectively.
Received 15 April 2011
Revised 8 June 2011
Accepted 12 June 2011
Available online 23 June 2011
Keywords:
Benzofuroxan derivatives
Topliss’ approach
MIC
Multidrug resistant Staphylococcus aureus
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
The Topliss decision tree considers the change in biological
activity for suggesting the next substituent group to be explored.
Staphylococcus aureus is notorious for its ability to become resis-
tant to antibiotics, and resistance turns it into an important hospi-
tal and communitarian pathogenic species of great concern.1,2
Since the introduction of penicillin in the 1940s, S. aureus has
developed resistance to all licensed antibacterial agents.3,4 The
selection of resistance is an inevitable consequence of the incorrect
or inappropriate antibiotic use, and to address this challenge the
search for novel antibiotics and/or new therapeutic strategies is
needed. In this regard, a major scientific community mobilization
has already begun for finding alternatives to treat these multidrug
resistant strains.5–9 Structure–activity relationship studies, which
consider the chemical structure of a certain compound and its abil-
ity to exhibit a desirable biological effect, could assuredly contrib-
ute to the development of new promising synthetic derivatives.10
Topliss method is usually employed to guide the choice of sub-
stituents in the support of drug design efforts toward the identifi-
cation of the most potent agents. This method was developed to
maximize the chances of synthesizing the most potent compounds
in the investigated set as early as possible. Furthermore, it was
based upon the assumption that the biological activity depends
on the hydrophobic and/or electronic effect of the substituent
groups present in the aromatic ring.11,12
The introduction of a chlorine (4-Cl), for example, led to a more po-
tent than a non-substituted compound, so a 3,4-Cl2 disubstituted
analogue should be synthesized. However, the decision of which
compound between the 3,4-Cl2 and 4-Cl analogues should be the
next one in the investigation process will depend on their biologi-
cal activity values. If the disubstituted compound presents the best
activity, then the diagram suggests that the next most active com-
pound would be the 4-NO2, 3-CF3 disubstituted derivative, consid-
ering the increment of both, the electronic (+r) and hydrophobic
(+ ) effects. Otherwise, if the activity values are higher than or
p
equal to the monosubstituted compound (4-Cl), the diagram will
follow until the 4-NO2 compound, suggesting the biological activ-
ity depends mainly upon the electronic effect. Coming back to the
4-Cl derivative, when the introduction of a chlorine led to an equi-
potent analogue in comparison to the parent compound, it can be
interpreted as +
the synthesis of a 4-CH3 analogue, which presents +
ues. Starting at the 4-CH3 derivative, new branches are suggested
to find the most active compounds. Finally, when the 4-Cl analogue
is less active than the non-substituted compound, it might imply in
the presence of steric hindrance, or the activity would be influ-
p
and ꢀ
r
effect. Then, the Topliss scheme suggests
and ꢀ val-
p
r
enced by ꢀ
p
or ꢀ
r effect. In this case, a 4-OCH3 substituted ana-
logue should be explored.11,12
Benzofuroxan (benzofurazan oxide; benzo[1,2-c]1,2,5-oxadiaz-
ole N-oxide) is an interesting ring system which has attracted
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.034

5032
S. D. Jorge et al. / Bioorg. Med. Chem. 19 (2011) 5031–5038
attention particularly due to its important biological activities,
which are being extensively reviewed and reported.13–15 Benzof-
uroxan derivatives have shown a wide spectrum of relevant biolog-
ical activities, such as antiprotozoal, antifungal, platelet
antiaggregatory and NO-releasing activity,16 suggesting these com-
pounds are quite promising for developing new potential lead
drugs.
Recent studies developed by our research group have demon-
strated that physicochemical properties as hydrophobicity (
p)
and electronic distribution ( ) can significantly influence the anti-
r
bacterial activity of benzofuroxan derivatives, which are nifuroxaz-
ide´s functional analogues.9,17 The mechanism of action of
nifuroxazide suggests that the antimicrobial activity of this com-
pound is related to the reduction of the nitro group and formation
of free radical toxic species.18 Based on this assumption, the deriv-
atives containing benzofuroxan as a pharmacophoric group would
act as nifuroxazide, possibly reducing the N-oxide group and caus-
ing similar effects.19–22
So, in this study, the purpose was the identification of the most
active compound against 3SP/R33 and VISA3 multidrug-resistant
strains of S. aureus in a set of benzofuroxan derivatives designed
by applying the Topliss’ decision tree. The optimization of the syn-
thetic pathway by reducing the synthetic steps and by improving
the yield of the final compounds was also considered as a key point
in this work.
Additionally, to explore the data related to the set of com-
pounds, particularly regarding the physicochemical properties of
the substituents chosen through the Topliss method and their
respective contributions on bioactivity, a hierarchical cluster anal-
ysis (HCA) was also performed. The primary purpose of HCA is to
present data in a manner which emphasizes natural groupings dis-
playing them in the form of a dendrogram, which allows the visu-
alization of such categories or clusters in a two-dimensional
space.23
Scheme 1. Synthesis of benzofuroxan derivatives. Reaction conditions: (i) CH3OH,
H2SO4/reflux; (ii) N2H4 64% aq/75 °C; (iii) CH3OH, H2SO4/reflux; N2H4 80% aq/rt; (iv)
5-formylbenzofuroxan, H2O, H2SO4, CH3COOH, C2H5OH/reflux.
The data considered to perform HCA, in this study, were the
Hammett (r, electronic contribution) and Hansch (p, hydrophobic
contribution) constant values of the substituents (R1 and R2) cho-
sen in the Topliss approach for the eight benzofuroxan derivatives.
Also, the van der Waals radii, which can express volume, were used
for the R3 substituent, since the ortho position in the aromatic ring
is related to the steric effect. Biological data correspond to the
dependent variable and their values were expressed as potency
(log 1/C) (see Table 1S in Supplementary data).
2. Methods
2.1. Synthesis
The designed compounds were obtained as shown in Scheme 1.
As already mentioned, the choice of the substituent groups was
based upon Topliss approach, aiming to obtain the most active com-
pound in relationship to the initial non-substituted compound. The
benzhydrazides used to obtain the final compounds were synthe-
sized employing two synthetic approaches. In the pathway A, the
substituted benzoic acids (1a–h) were converted into their methyl
benzoates (2a–h) by Fischer esterification. The ammonolysis of
intermediates 2a–h with hydrazine hydrate resulted in benzhyd-
razides 3a–h. Otherwise, in the pathway B, the benzhydrazides
(3a–h) were obtained directly from benzoic acids in one pot synthe-
sis, without the need of isolating their respective intermediate
esters (2a–h). Further, benzofuroxan derivatives (4a–h) were ob-
tained by reaction of the derivatives 3a–h with 5-formylbenzofuro-
xan. The aldehyde used in the final stage of the synthesis of
compounds 4a–h was synthesized from 4-chloro-3-nitrobenzalde-
hyde as reported in reference.24
HCA was carried out using the Pirouette 3.11 program,25
employing the complete linkage method and Euclidean distance.
The distances between samples or variables were calculated and
transformed into a similarity matrix whose elements correspond
to the similarity indexes. The similarity scale ranges from zero to
one, and the larger is the similarity index the smaller is the dis-
tance between any pair of samples or variables.23 The results will
be visualized as a dendrogram, which is a tree-shaped map con-
structed from the distances data.
Multivariate distance was computed on the independent vari-
able block, the transformed and preprocessed (autoscaling) matrix
X. The multivariate distance dab between two samples vectors, a
and b, was determined by computing differences at each of the
m variables:
M
1=M
dab ¼ ½
R
mðxaj ꢀ xbjÞ ꢁ
i
2.2. Hierarchical cluster analysis (HCA)
M is the order of the distance, and here corresponds to the
Euclidean distance (M = 2).25 Because inter-sample distances can
vary with the type and number of measurements, it is customary
to transform them onto a somewhat more standard scale of
similarity:
In HCA, distances between pairs of samples or variables are cal-
culated and compared. When distances between samples are rela-
tively small, this implies that the samples are similar, at least with
respect to the measurements in hand. Dissimilar samples will be
separated by relatively large distances. Thus, HCA groups data into
clusters having similar attributes, which is known in biological
sciences as numerical taxonomy.
dab
similarityab ¼ 1 ꢀ
dmax

S. D. Jorge et al. / Bioorg. Med. Chem. 19 (2011) 5031–5038
5033
The largest distance in the data set is dmax. As already men-
MIC values varied from 13.1 to 11.8
lg/mL (potency = 4.43, Table
tioned, on this scale, a value of 1 is assigned to identical samples
1), and the hydrophobic (p) and electronic (r) contributions also
increased because these physicochemical properties are additive
(R1 = R2 = Cl).
and a value of 0 to the most dissimilar samples.25
2.3. Biological activity evaluation
Following the selected branch, the 4-NO2, 3-CF3 disubstituted
compound (R1 = NO2,
p
4-NO2 = 0.24,
r4-NO2 = 0.78, R2 = CF3,
The minimum inhibitory concentration (MIC) method was used
to evaluate the antibacterial activity of the synthesized compounds
against 3SP/R33 and VISA3 multidrug-resistant strains of S. aureus
and the procedure was performed in two steps. In the first step,
called Phase I, the antibacterial activity was determined using
the classic method of serial broth microdilution.26 The second step,
Phase II, was used for maximizing the sensitivity of the test by lim-
itation of MIC values, which were defined in Phase I.9
p
3-CF3 = 1.21, 3-CF3 = 0.43, 4d) would be even more active. Then,
r
it was synthesized (Fig. 1). The intermediated compounds (2d ester
and 3d benzhydrazide) used to synthesize 4d compound are
unpublished, and their chemical structures were confirmed by 1H
and 13C NMR spectroscopy. Centesimal analysis was employed as
reference for testing the purity degree (details in Experimental Sec-
tion). The 4d compound, which showed a MIC value range from
12.7 to 11.4
series. The hydrophobic (
stituent groups have certainly presented influence on the antimi-
crobial effectiveness. Regarding the Hammett ( ) and Hansch (
constant values, the 4-NO2 substituent has a more relevant contri-
bution on the electronic effect ( 4-NO2 = 0.78) whereas the 3-CF3
substituent is more efficient on the hydrophobic feature
3-CF3 = 1.21). Anyway, as already mentioned, these physicochem-
l
g/mL (potency = 4.49), was the most active of this
p
+) and electron-withdrawing ( +) sub-
r
3. Results and discussion
r
p)
The Topliss operational scheme is used to provide a rational de-
sign of analogues, allowing the investigation of electronic, hydro-
phobic and even steric effects on bioactivity without the need of
synthesizing a large number of compounds (see Fig. 1). The yield
(%), melting point and MIC value ranges found for the benzofuro-
xan derivatives 4a–h are presented in Table 1.
r
(p
ical properties have an additive character. The summation of the
contributions of these substituents to the whole molecule is 1.75
and 1.21 regarding both, the hydrophobic (p) and electronic (r)
The initial compounds synthesized were the non-substituted
character, respectively. This balance of the hydrophobic-electronic
contribution could be considered quite suitable to the antimicro-
bial activity.
Regarding the Topliss’ decision tree, the best compound of the
investigated set was already found. However, to generate more
findings by exploring the Topliss diagram, observing the series
behavior, and optimizing synthetic pathways, some compounds
near to the right branch of the decision tree (see Fig. 1) whose
(R1 = H;
4-Cl = 0.70,
lue ranges from 18.0 to 16.2
17.0 to 15.3 g/mL (potency = 4.27), respectively (Table 1). The
higher potency showed by 4b suggest that the bioactivity improve-
ment can be related to the hydrophobic ( +) and/or electron-with-
drawing character ( +) of the substituent chlorine (R1).
p
= 0.0,
4-Cl = 0.23; 4b) compound, which presented MIC va-
g/mL (potency = 4.19) and from
r = 0.0; 4a) and 4-Cl substituted (R1 = Cl,
p
r
l
l
p
r
According to the Topliss diagram approach (see Fig. 1), the next
compound synthesized was 3,4-dichloro disubstituted (R1 = Cl,
activities would be
Thus, 4-CF3 substituted (R1 = CF3,
and 4-Br substituted (R1 = Br, 4-Br = 1.19,
pounds were synthesized and presented MIC value ranges from
14.6 to 13.1 g/mL (potency = 4.38), and from 20.0 to 18.0 g/mL
r
-dependent were also synthesized.
4-CF3 = 0.54, 4-CF3 = 1.07, 4e)
4-Br = 0.23; 4f) com-
p
r
p4-Cl = 0.70, r4-Cl = 0.23, R2 = Cl, p3-Cl = 0.76, r3-Cl = 0.37; 4c). This
p
r
l
l
(potency = 4.26) (Table 1), respectively. The bioactivity of 4f com-
pound is more influenced by the hydrophobic contribution of the
substituent.
After, a 2,4-dichloro disubstituted derivative (R1 = Cl,
4-Cl = 0.23, R2 = H, = 0.00, = 0.00, R3 = Cl, 4g) was synthesized
from the 3g intermediate compound, which was obtained following
pathway B (Scheme 1). The MIC values varied from 19.7 to 17.7 g/
p4-Cl = 0.70,
r
p
r
l
mL (potency = 4.25). The presence of 2-Cl instead of 2-H confers a
bigger steric hindrance on that region, since the ortho position is pri-
marily related to the steric effect of the substituent. This kind of
molecular modification seems to be not favorable to the bioactivity,
since the 4g compound is less potent than the 4c analogue (3,4-di-
chloro disubstituted) (see Table 1). Additionally, the potency of the
4g compound is similar to the monosubstituted compound (4-Cl),
4b. Regarding the synthetic procedure, the 2,4-methyl dic-
hlorobenzoate ester (2g) was not obtained by pathway A because
the purification steps have failed.
The 4-NO2 substituted compound (R1 = NO2,
4-NO2 = 0.78, 4h) was the last compound synthesized, and also
the less active in this set. The MIC value ranged from 24.3 to 21.9
g/mL (potency = 4.16) (Table 1). This data can indicate that the
value of 4-NO2 substituent is far from -optimum, since the antimi-
crobial potency decreased in comparison to the unsubstituted com-
pound (4a), and it was not compensated by the electronic effect ( ).
p4-NO2 = 0.24,
r
l
p
p
r
The biological data found for the derivatives in this branch of
the decision diagram indicated that when the substituent group
Figure 1. Sequence of the potential antibacterial benzofuroxan compounds
planned from non-substituted derivative 4a employing the Topliss method. Dashed
line indicates the most active sequence in this study. [Adapted with permission
from J. Med. Chem., 15, Topliss, J. G. Utilization of operational schemes for analog
synthesis in drug design, 1006–1011 Copyright (1972) American Chemical Society.]
presents
r and p values out of the considered optimum values,
the antimicrobial activity decreases in the following direction: 4e
>> 4f >> 4g >> 4h.

5034
S. D. Jorge et al. / Bioorg. Med. Chem. 19 (2011) 5031–5038
Table 1
Experimental data found for the synthesized benzofuroxan derivatives
R1
R2
R3
MW
Yield (%)
Melting point (°C)
MIC (
l
g/mL)a 3SP/R33b, VISA3c
Phase II
Cd (10ꢀ5) (M)
Potency log 1/Cd
Phase I
4a
4b
4c
4d
4e
4f
4g
4h
H
Cl
Cl
NO2
CF3
Br
Cl
NO2
H
H
Cl
CF3
H
H
H
H
H
H
H
H
H
H
Cl
H
282.08
316.04
350.00
395.05
350.06
359.99
350.00
327.06
94
91
91
73
82
90
73
94
214.0–215.0
212.0–213.0
205.0–206.0
191.0–192.0
201.0–202.0
210.0–211.0
163.0–164.0
262.0–263.0
20.0–10.0
20.0–10.0
15.0–7.5
18.0–16.2
17.0–15.3
13.1–11.8
12.7–11.4
14.6–13.1
20.0–18.0
19.7–17.7
24.3–21.9
6.38
5.38
3.74
3.21
4.17
5.56
5.62
7.43
4.19
4.27
4.43
4.49
4.38
4.26
4.25
4.16
15.0–7.5
20.0–10.0
20.0––10.0
30.0–15.0
30.0–15.0
Ampicilin
Vancomycin
32.0–16.0 (3SP/R33)
1.0–0.5 (3SP/R33)
32.0–16.0 (VISA3)
8.0–4.0 (VISA3)
a
b
c
Values corresponding to the average of triplicates.
Resistant to 19 antibiotics.
Vancomycin-intermediate Staphylococcus aureus strain.
d
Regarding the potency determination (log 1/C), the highest C value of phase II range was converted into molarity, which is based upon the compounds molecular weight
(MW).
HCA can focus on samples or variables. Clustering of samples re-
not show sinergic effect with the compounds tested, due to the
low solubility of the bezofuroxan derivatives into culture media.
Ampicilin and vancomicyn, frequently used in antimicrobial thera-
peutic, were used as standard drugs.
The MIC intervals determined were the same for the two multi-
drug-resistant strains of S. aureus. The two strains were chosen
based upon their multidrug resistance for nineteen antibiotics cur-
rently used in therapeutic to treat S. aureus infections. They differ
only in their vancomycin vulnerability.27,28 The 3SP/R33 strain is
veals similarities among the samples while clustering of variables
pinpoints intervariable relationships. In this study, HCA was
couched in terms of samples (compounds) clustering and the find-
ings are presented in Figure 2.
HCA showed that the designed compounds were grouped into
two main groups (see Fig. 2). The group A has the unsubstituted
compound (4a, similiarity index = 0.27) and two subgroups (A0
and A00), which correspond to the monosubstituted compounds.
The subgroup A0 is composed by the compounds whose bioactivi-
sensitive to vancomycin (MIC 6 2 lg/mL), and resistant to amoxi-
ties are mostly
r
-dependent (4h and 4f) whereas subgroup A00 con-
cyllin/clavulanic acid, ampicillin, cefazolin, cefotaxime, cefalotin,
ciprofloxacin, clindamicin, erythromycin, gentamicin, imipinem,
nitrofurantoin, norfloxacin, oxacilin, penicillin, rifampicin and tri-
methoprim/sulfamethoxazole. The VISA3 strain shows intermedi-
tains the compounds in which the substituents present a more
pronounced hydrophobic character (4g and 4b). The similarity in-
dexes found for the compounds in the subgroups A0 and A00 were
0.75 and 0.71, respectively. The group B contains the R1 and R2
disubstituted compounds, including the more active compound
(B0, 4d). The similarity index between 4d e 4c is 0.52 (subgroup
B0). Although the 4e compound is part of group B, its similarity in-
dex is 0.18.
ate resistance to vancomicyn (MIC P 4 lg/mL), besides the
antibiotics already cited.
The Clinical and Laboratory Standards Institute of United States
considers as susceptible to vancomycin those S. aureus strains that
present MIC values 6 2
g/mL and 8 g/mL, the strains are called as vancomycin inter-
mediate resistant (VISA), and the strains are considered resistant
to vancomycin (VRSA) when the MIC values are P 16 g/mL.
Vancomycin does not work for patients infected with S. aureus
strains for which the MIC values are P8
g/mL.26
l
g/mL.26,29 If the MIC values are between
The biological assays were performed in triplicate in two stages,
phase I and II, increasing the results’ reliability. Dimethyl sulfoxide
(DMSO) was used as solvent at minimal concentration, which did
4
l
l
l
l
Among the eight compounds synthesized and identified in this
study, there are four new chemical entities, 4c, 4d, 4g, 4h (experi-
mental section). The identification of compounds 4a, 4b, 4e and 4f
was carried out through the comparison of the experimental melt-
ing point values to those previously reported in the literature (see
Table 5S, in Supplementary data), and also using 1H and 13C NMR
spectroscopy.
The designed compounds were prepared employing two syn-
thetic pathways, A and B (Scheme 1). For the pathway A, the final
compounds were synthesized from the corresponding benzoic
acids in three steps: esterification, ammonolysis, and preparation
of Schiff’s bases. The esterification step provided satisfactory yields
for all compounds (81–96%). However, lower yields (51–90%) were
obtained from the ammonolysis step probably due to the differ-
ence of reactivity among methyl esters under the influence of
Figure 2. Dendrogram found for the eight antimicrobial agents (4a–h) applying
HCA. The cursor (dashed line) is pointing out a similarity index of 0.50.

S. D. Jorge et al. / Bioorg. Med. Chem. 19 (2011) 5031–5038
5035
Table 2
Global yields obtained in benzhydrazides synthesis by pathway A and B
R1
R2
R3
Pathway A (
g
%)
Pathway B (
g
%)
Ratio B:A (%)
Methyl esters
Benzhydrazides
Globala
3a
3b
3c
3d
3e
3f
H
Cl
Cl
NO2
CF3
Br
Cl
NO2
H
H
Cl
CF3
H
H
H
H
H
H
H
H
H
H
Cl
H
89
96
88
89
81
98
85b
96
67
88
90
66
59
82
88c
51
60
84
79
59
48
80
74
49
88
85
90
73
81
95
80
76
48
1
14
24
69
18
8
3g
3h
55
Average
90
74
67
84
22
a
b
c
g
% global =
g
% methyl esthers *
g
% benzhydrazides/102.
Ref. 31.
Ref. 32.
substituent groups as well as some difficulties associated to the
crystallization procedure. The identification of methyl esters and
benzhydrazides was performed through the comparison of the
experimental melting point values to those previously reported
in the literature (see Tables 2S and 3S in Supplementary data).
As a trial to improve the yield, benzhydrazide synthesis was
developed in one step pot (pathway B, Scheme 1). The esters disso-
lution into ethanol,30 skipping the isolation step after esterification,
simplified the amonolysis reaction. For this reaction, hydrazine hy-
drate was added to the system. In the first step, the alcohol and acid
contributed favorably to the ester carbon protonation, becoming it
more susceptible to the hydrazine nucleophilic attack, thus, the
reaction was driven to the formation of benzhydrazides.
Benzhydrazides were identified through the melting point pro-
cedure and 1H and 13C NMR spectroscopy. The findings were com-
pared to those previously reported in the literature (see Table 4S in
Supplementary Material). The average yield found for this syn-
thetic pathway was 84% (73–95%), and the data are presented in
Table 2. For comparison, the information regarding the conditions
used to obtain the compounds methyl 2,4-dichlorobenzoate 2g and
2,4-dichlorobenzhydrazide 3g are from the literature.31,32 The one
pot synthesis showed greater yield values in comparison to a mul-
ti-step synthesis, and indeed can be consider as a better synthetic
pathway.
The synthesis methodology was modified to obtain 4-nitro-3-
(trifluoromethyl)benzhydrazide, 3d and 4-nitrobenzhydrazide 3h.
Hydrazine hydrate acts as reducing agent, which is widely used
in the reduction of nitro aromatic groups.33 The reduction process
is favoured by the use of catalysts, such as, metallic ferrous, iron
(III) hydroxide, or graphite.34,35 However, despite the time of reac-
tion used in this stage has not been so long, the convertion through
reduction of the nitro group to amino in the aromatic ring ocurred
for the both intermediates. This fact was confirmed by 1H NMR
spectra (see Fig. 3), considering the chemical deviation (d) related
to the internal standard reference (tetrametilsilane). It was ob-
served a singlet with two protons integration at d 6.0 ppm (2d)
and d 5.3 ppm (2h) regions, respectively, indicating the presence
of NH2 group at para position in the aromatic ring.36 One alterna-
tive to avoid the reduction of these compounds is to perform the
reactions in a controlled temperature of -3 °C and 2 °C.
Figure 3. 1H NMR spectra of the isolated products when different temperatures
were employed in the ammonolysis of compound 2h (using pathway A) or 1h
(using the pathway B).
medium using a simple filtration step, avoiding an additional
recrystallization procedure. It is noteworthy that the differences
in the structure of the crystals depend upon the temperature in
which the system is maintained after reaction. The crystals habits
tend to be larger when the system is kept at room temperature
than at temperatures below 0 °C.
Finally, the classic method of Schiff’s base preparation was em-
ployed as the last step reaction. This reaction is easy to perform,
and the resulting yields were around 86% (73%–94%). The partial
and total yields obtained by the pathways A and B are presented
in Table 3. The synthesis of benzofuroxan derivatives was per-
formed in three steps using the pathway A (Scheme 1), and the
average global yield was 57%. Otherwise, when the eight
The products obtained by pathway B (Scheme 1) presented a
crystal structure and they could be separated from the reaction

5036
S. D. Jorge et al. / Bioorg. Med. Chem. 19 (2011) 5031–5038
Table 3
Global yields obtained in pathway A and B
R1
R2
R3
Pathway A (
g
%)
Schiff’s bases
Pathway B (
g
%)
Ratio B:A (%)
Methyl esters
Benzhydrazides
Globala
Benzhydrazides
Schiff’s bases
Globalb
4a
4b
4c
4d
4e
4f
4g
4h
H
Cl
Cl
NO2
CF3
Br
Cl
NO2
H
H
Cl
CF3
H
H
H
H
H
H
H
H
H
H
Cl
H
89
96
88
89
81
98
85c
96
90
67
88
90
66
59
82
88d
51
74
94
91
91
73
73
82
90
94
86
56
77
72
43
35
66
67
46
57
88
85
90
73
81
95
80
76
84
94
91
91
73
73
82
90
94
86
83
77
82
53
59
78
72
71
72
48
1
14
24
69
18
7
55
25
Average
a
b
c
g
g
% global =
% global =
g
g
% methyl esthers *
% benzhydrazides *
g
g
% benzhydrazides *
% Schiff’s bases/102.
g
% Schiff’s bases/104.
Ref. 31.
Ref. 32.
d
compounds were synthesized using pathway B (Scheme 1) the
average global yield obtained was 72%, meaning a gain around
25% in comparison to the pathway A. Thus, to increase the global
yield, the ammonolysis reaction was performed subsequently to
esterification, skipping the ester isolation step.
The compound that significantly showed the efficiency of syn-
thesis optimization was the 4-trifluoromethyl-[N0-(benzofuroxan-
5-yl)methylene]benzhydrazide, 4e. When three synthetic steps
were used the global yield was 35% whereas when just two syn-
thetic steps were employed the global yield raised to 59%, meaning
a real yield improvement of 70%. Then, the exclusion of one syn-
thetic step can also reflect in economy of time, quantity of raw
material and solvent.
using Micro-Química MQAPF-301 apparatus and elemental analy-
sis was performed on a Perkin-Elmer 24013 CHN Elemental
Analyzer.
5.1.1. General procedure for the preparation of methyl esters (2)
Each substituted benzoic acid 1a–h (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 was washed with cold water.
5.1.1.1. 4-Nitro-3-(trifluoromethyl)benzoic acid methyl ester
(2d). Pale white needle crystal (89%); mp 68.0–69.0 °C. 1H NMR
(DMSO-d6, 300 MHz): d (ppm): 8.46–8.41 (m, 1H, H6), 8.35 (s,
1H, H2), 8.31–8.25 (m, 1H, H5), 3.96 (s, 3H, CH3). 13C NMR {H}
(DMSO-d6, 75 MHz): 164.9 (C@O), 149.9 (C4), 135.5 (C1), 134.1
(C6), 128.7 (C2), 126.5 (C5), 122.3 (C3), 121.8 (CF3), 53.5 (CH3).
Anal. Calcd for (C9H6F3NO4) C, H, N: calc: C, 43.39; H, 2.43; N,
5.62. exp: C, 43.26; H, 2.48; N, 5.59.
4. Conclusions
In this study, the synthesis, identification, and biological assay
of a set of benzofuroxan derivatives was explored. The choice of
substituent groups was based upon the Topliss approach, which
has indicated as more active compound of the set, the 4-nitro-3-
trifluoromethyl-[N0-(benzofuroxan-5-yl)methylene]benzhydrazide
5.1.2. General procedure for the preparation of benzhydrazides
(3)
(4d), showing a MIC value 6 12.7
resistant S. aureus strains.
lg/mL against the multidrug-
Pathway A—Hydrazine hydrate 64% (v/v) (30.0 mL, 0.33 mol)
was heated up to 50–60 °C. The methyl ester previously isolated
(0.01 mol) was added and the mixture was refluxed during
10 min. The cooling down was proceeded sequentially in a water
bath, followed by ice bath and dry ice - ethanol bath. The solid
was filtered and washed with cold water. Different conditions were
needed to obtain 4-nitro-3-(trifluoromethyl)benzhydrazide (3d)
and 4-nitrobenzhydrazide (3 h). Hydrazine hydrate 64% (v/v)
(30.0 mL, 0.33 mol) was cooled down in ice bath to ꢀ3 to 2 °C.
The respective methyl ester (0.01 mol) was added and the mixture
was stirred during 1 hour. The cooling down was proceeded in dry
ice – ethanol bath. The solid was filtered and washed with cold
water. Pathway B—each substituted benzoic acid (0.01 mol) was re-
fluxed during 4 h in 20.0 mL (0.50 mol) of anhydrous methanol and
0.5 mL (1.0 mmol) of sulfuric acid. The reaction mixture was cooled
down to room temperature. and the hydrazine hydrate 80% (v/v)
(10.0 mL, 0.11 mol) was added. The system was maintained into
vigorously stirring for more 30 minutes. In the case of compounds
with 4-nitro and 4-nitro-3-trifluoromethyl substituent groups at-
tached in the benzene moiety, after the addition of hydrazine hy-
HCA divided the designed compounds in two main groups
according to the substitution patterns, and the subgroups regard-
ing the influence of the electronic and hydrophobic character of
the substituent on bioactivity.
All synthesis reactions have presented satisfactory yields, and
the melting point determination procedures have attested the high
purity degree of the resulting compounds. Additionally, the one-
step synthesis of benzhydrazides was considered more efficient
than the same synthesis using two steps, besides to be an easier
method and less time consuming.
5. Experimental
5.1. Chemistry
NMR spectra were recorded on a Bruker ADPX Advanced
(300 MHz) spectrometer employing DMSO-d6 solutions with tetra-
methylsilane as internal standard. Melting points were determined

S. D. Jorge et al. / Bioorg. Med. Chem. 19 (2011) 5031–5038
5037
drate 80% (v/v) at room temperature, the reaction mixture was
cooled down in ice bath and maintained into stirring during 1 hour.
After these periods, the mixture was maintained at cold tempera-
ture to give 3.
(C14, C16), 116.7 (C7), 115.6 (C4); Anal. Calcd for (C14H9N5O5): C,
51.38; H, 2.77; N, 21.40. Found: C, 51.60; H, 2.79; N, 21.00.
5.2. Biology
5.1.2.1. 4-Nitro-3-(trifluoromethyl)benzhydrazide (3d). White so-
lid (pathway A—66%; pathway B—73%); mp 166.0–167.0 °C. 1H
NMR (DMSO-d6, 300 MHz): d (ppm): 10.25 (s, 1H, NH), 8.33 (s,
1H, H2), 8.30 (d, 1H, J(6,5) = 8.3 Hz, H6), 8.21 (d, 1H, J(5,6) = 8.3 Hz,
H5), 4.66 (s, 2H, NH2). 13C NMR {H} (DMSO-d6, 75 MHz): d
(ppm): 162.8 (C@O), 148.8 (C4), 137.7 (C1), 133.3 (C6), 127.0
(C2), 126.2 (C5), 122.1 (C3), 121.7 (CF3); Anal. Calcd for
(C8H6F3N3O3) C, H, N: calc: C, 38.57; H, 2.43; N, 16.87. exp: C,
38.67; H, 2.30; N, 16.87.
Phase I—Minimal Inhibitory Concentration (MIC) of the com-
pounds was determined with 96-well microliter plates containing
two-fold serial dilutions of the compounds in Tryptic Soy Broth
(TSB-SigmaÒ) medium. Stock solutions of the compounds were
prepared in DMSO/TSB 1:10 v/v. Concentrations ranged from 0.1
to 80 lg/mL, using ampicilin 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 during 18 h. The lowest concentration of compound—that
in which there was no visible growth—was considered as the
MIC value. Readings at 24 and 48 h were carried out for sterility
control. Experiments were performed in triplicate.
Phase II—Stock solutions (1 mL) were prepared using two-fold
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 column.
For the positive growth control, 0.1 mL of TSB was added to column
12. Bacterial suspensions were prepared by the same procedure
described in phase I, and 0.1 mL of inoculum was transferred to
each column, except to the column 11. The plates were mixed
and incubated at 35 °C during 18 h.
5.1.3. General procedure for the preparation of benzofuroxans
derivatives (4a–h)
A mixture of 5-formylbenzofuroxan (1.0 mmol) and benzhyd-
razides (1.0 mmol) in water, sulfuric acid, acetic acid and methanol
(8:7:8:20 v/v) was heated under reflux during 1 h. After cooling
down, the mixture was poured into cold water to give 4.
5.1.3.1. 3,4-Dichloro-[N0-(benzofuroxan-5-yl)methylene]benzh-
ydrazide (4c). Yellow solid (91%); mp 205.0–206.0 °C. 1H NMR
(DMSO-d6, 300 MHz): d (ppm): 11.96 (s, 1H, H10), 8.36 (s, 1H,
H8), 8.01 (d, 1H, J(13,17) = 1.5 Hz, H13), 7.79 (d, 1H, J(17,16) = 8.3 Hz,
H17), 7.77 (d, 1H, J = 9.4 Hz, H6), 7.73 (s, 1H, H4), 7.65 (d, 1H,
J(16,17) = 8.3 Hz, H16), 7.58 (d, 1H, J = 9.4 Hz, H7); 13C NMR {H}
(DMSO-d6, 75 MHz): d (ppm): 162.1 (C11), 145.9 (C8), 137.9 (C5),
137.8 (C15), 135.2 (C12), 134.0 (C14), 131.9 (C16), 131.6 (C13),
130.3 (C7a), 130.2 (C17), 129.3 (C3a), 128.6 (C6), 116.7 (C7),
115.5 (C4); Anal. Calcd for (C14H8Cl2N4O3): C, 47.89; H, 2.30; N,
15.96. Found: C, 47.87; H, 1.72; N, 15.56.
Acknowledgments
The authors thank Professor Elza Masae Mamizuka, from the
Department of Clinical and Toxicological Analysis-USP, for provid-
ing the 3SP/R33 and VISA3 Staphylococcus aureus strains; and the
Brazilian scientific committees CNPq, CAPES, and FAPESP for the
financial support.
5.1.3.2.
4-Nitro-3-(trifluoromethyl)-[N0-(benzofuroxan-5-
yl)methylene]benzhydrazide (4d). Yellow solid (73%); mp
191.0–192.0 °C. 1H NMR (DMSO-d6, 300 MHz): d (ppm): 12.20 (s,
1H, H10), 8.38 (s, 1H, H8), 8.34 (s, 1H, H13), 8.31 (d, 1H,
J = 9.2 Hz, H6), 8.18 (d, 1H, J(16/17) = 8.1 Hz, H16), 7.79 (s, 1H, H4),
7.71 (d, 1H, J(17/16) = 8.3 Hz, H17), 7.60 (d, 1H, J = 9.4 Hz, H7); 13C
NMR {H} (DMSO-d6, 75 MHz): d (ppm): 163.7 (C11), 149.3 (C15),
147.3 (C8), 137.7 (C12), 137.3 (C5), 133.5 (C17), 130.5 (C7a),
129.7 (C3a), 129.3 (C6), 128.3 (C13), 127.0 (C16), 122.6 (C14),
121.8 (CF3), 117.4 (C7), 114.4 (C4); Anal. Calcd for (C15H8F3N5O5):
C, 45.58; H, 2.04; N, 17.71. Found: C, 45.71; H, 1.98; N, 17.32.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.034.
References and notes
5.1.3.3. 2,4-Dichloro-[N0-(benzofuroxan-5-yl)methylene]benz-
hydrazide (4g). Yellow solid (73%); mp 163.0–164.0 °C. 1H NMR
(DMSO-d6, 300 MHz): d (ppm): 12.34 (s, 1H, H10), 8.35 (s, 1H,
H8), 8.15 (s, 1H, H14), 7.96 (s, 1H, H4), 7.86 (d, 1H, J = 9.2 Hz,
H6), 7.76 (d, 1H, J = 9.2 Hz, H7), 7.66 (d, 1H, J(17,16) = 8.1 Hz, H17),
7.60–7.52 (m, 1H, H16); 13C NMR {H} (DMSO-d6, 75 MHz): d
(ppm): 162.4 (C11), 146.8 (C8), 135.9 (C13), 135.0 (C5), 134.1
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for (C14H8Cl2N4O3): C, 47.89; H, 2.30; N, 15.96. Found: C, 47.56;
H, 1.65; N, 15.39.
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