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reducing the treatment options to the use of polimyxins (e.g.,
Colistin) and Tigecycline.8–21 The resistance of Gram-negative
bacteria strains to Tigecycline has already been reported.9,10
In addition, cases of sepsis especially associated with fungal
infections can also be a serious concern for healthcare systems.
The number of fungal infections has been increased more than
200% in the last two decades, being the third most common cause
of bloodstream infections.11,12 Candida spp. is responsible for 75%
of all nosocomial infections caused by fungus, and Candida albicans
causes 45% of the total cases primarily in immunosuppressed
patients.13 The treatment available comprises polyenes (Ampho-
terecin B), azoles (Fluconazole and Itraconazole), and, more
recently, echinocandins. However, similar to bacteria, several
strains have already presented resistance to those drugs.14,15
In this regard, it is important to adopt urgent measures to
prevent the inappropriate impact caused by multidrug-resistant
microorganisms on healthcare systems. Among this measures are
the correct prescription of antibiotics; the development and imple-
mentation of protocols for cleaning and disinfecting patient rooms,
surfaces, equipment, and common areas in hospitals environ-
ments; and, also the discovery of new drugs capable of treating
the multidrug-resistant bacterial infections.
Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter clocae,
Escherichia coli, Serratia marcescens, and Staphylococcus aureus). In
addition, derivatives that have showed promising activity in S. aur-
eus were evaluated against multidrug-resistant S. aureus VISA3.
The molecular properties were investigated by applying explor-
atory data analysis, a chemometric procedure, which comprises
the principal component analysis (PCA) and hierarchical cluster
analysis (HCA).31,32
2. Results and discussion
2.1. Chemistry
The designed compounds were obtained as show in Scheme 1.
The compounds of series I (substituted-((5-nitrofuran-2-yl)methy-
lene)benzohydrazide, 2a–v) were obtained from the reaction of
substituted benzohydrazides (1a–v) with 5-nitrofuran-2-carbalde-
hyde.33 The compounds of series II (3-acetyl-5-(substituted-phe-
nyl)-2-(5-nitro-furan-2-yl)-2,3-dihydro-1,3,4-oxadiazole,
3a–v)
were obtained by cyclization reaction of 2a–v with acetic anhy-
dride.29,34,35 The substituent groups attached at benzene moiety
were chosen based on their physicochemical properties, such as
hydrophobicity and electronic effect.36
Nitrofurans are a class of nitro compounds, which have been
used to treat bacterial infections since 1940s.16 Several studies
involving this chemical class have been carried out regarding
different therapeutic uses, such as tuberculostatic,17,18 antileish-
manial,19,20 trypanocidal activity,21 and anti-proliferative effect
on cancer cells lines.22 Nifuroxazide (NF), for instance, is a nitro
compound, which was widely used as antibacterial drug in 1970s
and 80s. Recently, NF has been reported as a potential Quorum-
Sensing inhibitor on Pseudomonas aerugionosa, even though it
seems not be able to stop the bacterial growth.23 NF is also consid-
ered an excellent lead compound due to its chemical structure,
which benefits molecular modifications by rational design
strategy. One of these modifications is the synthesis of 3-acetyl-
2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazole ring system by
cyclization reaction of N-acylhydrazone compounds, which was
reviewed by Rollas and Karakusß.24 Briefly, these structures were
investigated as monoamine oxidase inhibitors,25 antifungals,26
anticonvulsants,27 anti-inflammatory agents,28 antibacterici-
dals,29,30 and trypanocidal agents.29 The findings have emphasized
the potential of these molecular structures for the development of
novel drugs.
In this study, 41 compounds (22 compounds of series I and 19
compounds of series II) were synthesized and identified. All com-
pounds were synthesized in two steps, starting from the corre-
sponding benzohydrazides. The first step was based on classical
Schiff’s base formation, whose synthesis and mechanism of
reaction have been well reported and discussed.29,37 Satisfactory
yields (around 90%) were obtained in this step. The second step
was performed by a cyclization reaction of Schiff’s base, and pre-
sented 66% yield.35 The compounds were structurally identified
(see the Supplementary information section, p. S2–S49).
Compounds 3h (R1 = SO2NH2), 3j (R1 = N(CH3)2, 3k (R1 = NH2),
and the respective oxadiazole analogue of NF (R1 = OH) were not
obtained probably due to a reaction of the substituent groups with
acetic anhydride. The presence of unbound electrons seems to pro-
vide stronger nucleophiles, as the amidic nitrogen from Schiff’s
base, for instance. This observation was confirmed by 1H NMR
and 13C NMR spectra (Fig. 2), considering the chemical deviation
(d) related to the internal standard reference (tetrametilsilane).
Regarding the 1H NMR spectra of NF (Fig. 2A), it can be noticed
the presence of a singlet signal around d 12 ppm, which is related
to the proton of amidic nitrogen (H8). Also, a singlet signal at d
8.40 ppm indicated the azomethine hydrogen atom (H6). After a
cyclization reaction, the absence of the signal corresponding to
the amidic nitrogen and a singlet signal at d 7.35 ppm (H2), which
indicates the 2,3-dihydro-1,3,4-oxadiazoline ring group formation,
were observed in 1H NMR spectra of the product (Fig. 2B). Further-
more, a singlet with six protons integration at d 2.29 ppm was
observed, confirming the three protons related to the acetyl group
and to the acetoxylation of hydroxyl group. Analyzing the 13C NMR
spectra (Fig. 2C), there are signals at 167.1 and 20.9 ppm, which
indicate the presence of carbonyl (C20) and alkyl groups (C22),
respectively. Also, the signals at 153.4 (C5) and 84.6 ppm (C2)
are related to the oxadiazole ring system. The presence of the acet-
oxy group attached to benzene moiety can be observed through
the signals at 168.5 and 20.7 ppm.
Herein, two series of compounds structurally analogous of NF
(Fig. 1A) were designed, synthesized, and experimentally tested.
Series I presents a N-acylhydrazone structure (azomethine deriva-
tives), and series II has a heterocyclic ring system 3-acetyl-2,5-
disubstituted-2,3-dihydro-1,3,4-oxadiazole (oxadiazoline series)
(see Fig. 1B). Antimicrobial activity was evaluated against
microorganisms reported as HCAI pathogens (Candida albicans,
2.2. Biological activity
The minimal inhibitory concentration (MIC) was determined by
broth microdilution method against the following strains: Candida
albicans 537Y, Klebsiella pneumoniae ATCC 700603, Enterococcus
faecalis ATCC 29212, Enterobacter clocae ATCC 23355, Escherichia
coli ATCC 25922, Serratia marcescens ATCC 14576, and
Figure 1. (A) Chemical structure of nifuroxazide (NF), pointing out the regions
where molecular modifications were carried out on this study. (B) General chemical
structures of series I and II.
Zorzi, Rodrigo Rocha
