Synthesis, molecular properties prediction, and anti-staphylococcal activity of N-acylhydrazones and new 1,3,4-oxadiazole derivatives

Article abstract of DOI:10.3390/molecules17055095

Five new 1-(2-(5-nitrofuran-2-yl)-5-(aryl)_-1,3,4-oxadiazol-3- (2H)-yl) ethanone compounds 5a-e were synthesized by cyclization of N-acylhydrazones 4a-e with acetic anhydride under reflux conditions. Their structures were fully characterized by IR, 1H-NMR, and ¹³C-NMR. Furthermore, evaluations of the antibacterial activity of the 1,3,4-oxadiazoles 5a-e and N-acylhydrazones 4a-e showed strong activity against several strains of Staphylococcus aureus, with MICs between 4 μg/mL to 32 μg/mL. In silico studies of the parameters of Lipinski's Rule of Five, as well as the topological polar surface area (TPSA), absorption percentage (% ABS), drug likeness and drug score indicate that these compounds, especially 4a and 5d, have potential to be new drug candidates

Full text of DOI:10.3390/molecules17055095

Molecules 2012, 17, 5095-5107; doi:10.3390/molecules17055095
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Molecular Properties Prediction, and
Anti-staphylococcal Activity of N-Acylhydrazones
and New 1,3,4-Oxadiazole Derivatives
Cledualdo Soares de Oliveira 1, Bruno Freitas Lira 1, Vivyanne dos Santos Falcão-Silva 2,
Jose Pinto Siqueira-Junior 2, Jose Maria Barbosa-Filho 3
and Petronio Filgueiras de Athayde-Filho 1,*
1
Department of Chemistry, Federal University of Paraiba, João Pessoa, PB 58059-900, Brazil;
E-Mails: aldoscarchi@yahoo.com.br (C.S.O.); brunofrlira@hotmail.com (B.F.L.)
2
Department of Molecular Biology, Federal University of Paraíba, João Pessoa, PB 58059-900,
Brazil; E-Mails: vivyannefalcao@yahoo.com.br (V.S.F.-S.); jpsiq@uol.com.br (J.P.S.-J.)
3
Laboratory of Pharmaceutical Technology, Federal University of Paraíba,
João Pessoa, PB 58051-900, Brazil; E-Mail: jbarbosa@ltf.ufpb.br
* Author to whom correspondence should be addressed; athayde-filho@quimica.ufpb.br;
Tel.: +55-83-3216-7937.
Received: 19 March 2012; in revised form: 18 April 2012 / Accepted: 19 April 2012 /
Published: 3 May 2012
Abstract: Five new 1-(2-(5-nitrofuran-2-yl)-5-(aryl)-1,3,4-oxadiazol-3-(2H)-yl) ethanone
compounds 5a–e were synthesized by cyclization of N-acylhydrazones 4a–e with acetic
anhydride under reflux conditions. Their structures were fully characterized by IR,
1H-NMR, and 13C-NMR. Furthermore, evaluations of the antibacterial activity of the
1,3,4-oxadiazoles 5a–e and N-acylhydrazones 4a–e showed strong activity against several
strains of Staphylococcus aureus, with MICs between 4 μg/mL to 32 μg/mL. In silico
studies of the parameters of Lipinski’s Rule of Five, as well as the topological polar surface
area (TPSA), absorption percentage (% ABS), drug likeness and drug score indicate that
these compounds, especially 4a and 5d, have potential to be new drug candidates.
Keywords: 1,3,4-oxadiazole; N-acylhydrazone; Staphylococcus aureus; drug-likeness

Molecules 2012, 17
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1. Introduction
The introduction of antibiotics in the chemotherapy of bacterial infections in the middle of the last
century revolutionized medicine, causing a drastic reduction in mortality from bacterial diseases. However,
the spread and misuse of antibiotics has unfortunately helped the emergence of bacterial resistance,
whereby bacteria populations have developed defense mechanisms against most antibiotics [1–3].
Staphylococcus aureus is often cited as the principal hospital pathogen that is becoming
increasingly virulent and resistant to antibiotics, and the primary disseminator of human infectious
diseases around the World [4]. The ease with which S. aureus acquires resistance to virtually all
antibiotics currently in use is a matter of considerable concern [5,6].
Among compounds that possess a broad spectrum of biological activities are the nitrofurans. These
compounds were the first nitroheterocyclic used in chemotherapy. Three of them, furazolidone (1),
nitrofurantoin (2) and nitrofurazone (3) have been used to treat bacterial infections for more than
50 years [7,8]. Some derivatives of 5-nitrofuran, besides having antibacterial and antifungal properties,
exhibit antiprotozoal activity as is the case of nifurtimox (4), used in the treatment of trypanosomiasis,
and leishmaniasis [9,10]. Recently, it has also been reported that heterocyclic mesoionic compounds
(9) containing the 5-nitrofuran group modulate bacterial resistance, putatively acting as efflux pump
inhibitors [11], (Figure 1).
Figure 1. Chemical structures of biologically active molecules containing the 5-nitrofuran
groups and/or 1,3,4-oxadiazole: furazolidone (1), nitrofurantoin (2), nitrofurazone (3),
nifurtimox (4), raltegravir (5), nesapidil (6), furamisole (7), tiodazosin (8) and MC-2 (9).
O
O
O
N
NH
O
NH2
N
N
N
O
N
N
O2N
O
H
O
O
O2N
O
O2N
(1)
(2)
(3)
O
S
OH
F
N
O
H
N N
O
H
N
N
O
N
N
N
O2N
O
O
(4)
(5)
O
N
N
O
N
O
N N
NH2
N
O
OH
O
O2N
O
(6)
NH2
(7)
Cl
MeO
MeO
N
N N
S
N
N
N
N
N
S
N
O
H
O
(8)
O2N
O
(9)

Molecules 2012, 17
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Another important group of heterocyclic compounds for medicinal chemistry are the 1,3,4-oxadiazoles
which have been extensively reported in the literature for the synthesis of new biologically active
molecules with antibacterial [12–15], antifungal [16,17], analgesic [18], anti-inflammatory [19],
antiviral [20,21], antitumor [22,23], antihypertensive [24], enzyme inhibition [25,26] and
anticonvulsant [27,28] activities. Examples of drugs containing the 1,3,4-oxadiazole unit currently
used in clinical medicine are: raltegravir® (5), an antiretroviral drug [29], nesapidil® (6) an anti-arrhythmic
therapy [30], furamizole® (7) a nitrofuran derivative that has strong antibacterial activity [31] and
tiodazosin® (8) an antihypertensive drug [32] (Figure 1).
Therefore, considering that the 5-nitrofuran and 1,3,4-oxadiazole are important building blocks for
the development of new drug candidates, we synthesized the intermediate N-acylhydrazones 4a–e and
the target 1,3,4-oxadiazoline molecules 5a–e in order to evaluate their potential against Staphylococcus
aureus resistant to drugs, as well as to investigate the theoretical potential of these compounds to
become new drug candidates through in silico studies involving Lipinski’s Rule of Five, topological
polar surface area (TPSA), absorption percentage (% ABS), drug-likeness and drug score.
2. Results and Discussion
2.1. Chemistry
The synthesis of the new 1,3,4-oxadiazoline compounds 5a–e was carried out according to
procedures reported in the literature and the four synthetic stages are outlined in Scheme 1.
Scheme 1. Synthetic route for the synthesis of the target molecules.
O
O
O
MeOH
MeOH
NH2
OH
O
N
H
N2H4
reflux
H2SO4
reflux
R
R
R
O
3a-e
2a-e
1a-e
EtOH
AAG
reflux
H
O2N
O
O
14
10
3
2
4
13
8
O
N
N
3
4
7
N
11
Ac2O
reflux
2
NO2
10
12
O
1
N
H
8
5
7
O
9
6
R
O
1
5
12 NO2
15
6
R
13
14
4a-e
5a-e
R = H, Me, NO2, Cl, OMe
In step 1, the aromatic esters 2a–e were obtained in quantitative yields by Fischer esterification of
the respective aromatic carboxylic acids 1a–e. In step 2, hydrazinolysis of the esters 2a–e with
hydrazine hydrate (80%) provided aroylhydrazides 3a–e in excellent yields. Step 3 gave
N-acylhydrazones 4a–e in high yields and purity by condensation reaction between the respective

Molecules 2012, 17
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aroylhydrazides 3a–e and 5-nitro-2-furaldehyde in ethanol (95%) with glacial acetic acid as the
catalyst [33].
Heterocyclic compounds of the 1,3,4-oxadiazoline class can be easily obtained by reacting
N-acylhydrazones and acetic anhydride in excess with reflux [23,34,35]. The reaction of the
N-acylhydrazones 4a–e with acetic anhydride, Step 4, provided the compounds 5a–e in moderate
to good yields. The purity of compounds was verified examining their melting ranges and by
gas chromatography. The structures of final and intermediate products were fully confirmed by IR,
1H-NMR and 13C-NMR spectroscopic techniques. The 1H-NMR, and 13C-NMR spectroscopy data was
sufficient to confirm the formation of the 1,3,4-oxadiazole ring, as these compounds have very
1
characteristic signals. Thus, in the H-NMR spectra of compounds 5a–e two typical signals were
observed, one assigned to the methyl protons (H-14) in the aliphatic region of (2.2 to 2.4 ppm) and
another assigned to methinic protons (H-8) in the aromatic region (7.3 to 7.4 ppm), whereas for what
was not observed, the typical signals for the NH (H-8), and CH=N (H-6) seen in the 1H-NMR spectra
of compounds 4a–e were no longer visible. In the 13C-NMR spectra, signals characteristic of C=O
around 167 ppm, and alkyl around 23 ppm, as well as the oxadiazole ring signal (C-8) around 85 ppm
and (C-7) about 153 ppm were observed, thus confirming the formation of the 1,3,4-oxadiazole ring. In
the infrared spectrum, all the compounds 5a–e showed amide C=O absorption bands around 1,670 cm−1,
and C-O-C absorption stretches (oxadiazole ring) around 1,240 cm−1, and C=N absorptions
(oxadiazole ring) at around 1,635 cm−1.
2.2. In Silico Study of Molecular Properties and Drug-Likeness
Molecular properties such as membrane permeability and oral bioavailability are usually associated
with some basic molecular descriptors, such as log P (partition coefficient), molecular weight (MW),
and the acceptors and donor for hydrogen bonding in a molecule. Using these molecular properties,
Lipinski [36] established a controversial rule for drug design. Created in 1995 and published in 1997, it
is known as the “Lipinski rule” or “rule of five”, it has this name, because each of the four parameters
involved uses values that are multiples of five. The rule states that the compounds are more likely to be
orally bioavailable if they obey the following criteria: log p ≤ 5, molecular weight ≤500, hydrogen
bond acceptors ≤10, and hydrogen bond donors ≤5. Molecules that violate more than one of these rules
may have problems with bioavailability. Therefore, this rule establishes some structural parameters
relevant to the theoretical prediction of the oral bioavailability profile, and is widely used in designing
new drugs. However, classes of compounds that are substrates for biological transporters such as
antibiotics, antifungals, vitamins, and cardiac glycosides, are exceptions to the rule [36].
As such, we decided to carry out studies in silico for Lipinski parameters, as well as in the
topological polar surface area (TPSA), the percentage of absorption (% ABS) and we include the
drug-likeness and drug score (representing the combined physicochemical, pharmacokinetic and
pharmacodynamic effects of a compound) for the N-acylhydrazone compounds 4a–e and the
1,3,4-oxadiazoles 5a–e in order to verify that these compounds exhibit good (theoretical) oral
bioavailability potential.
The lipophilicity (log P) and topological polar surface area (TPSA) were calculated using the online
software Molinspiration [37], while the aqueous solubility, drug-likeness and drug score were

Molecules 2012, 17
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calculated using the OSIRIS property explorer software [38]. For the study of drug-likeness, the
OSIRIS program uses a list of 5,300 molecular fragments, where the frequency of occurrence of each
fragment is determined based on a collection of 3,300 drugs and 15,000 commercially available
chemicals (Fluka) that are not drugs [38]. The percentage of absorption was estimated using the
Equation: % ABS = 109 − (0.345 × TPSA), according to Zhao et al. [39]. The calculations data are
shown in Table 1.
Table 1. Calculated absorption (% ABS), polar surface area (PSA), LogS, Lipinski
parameters, drug-likeness and drug score of the compounds 4a–e and 5a–e.
Lipinski’s Parameters
TPSA
(Ų)
%
Log Drug-
Drug
Comp. HBA HBD MW miLogP Violations
ABS
S
likeness score
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
7
7
1
1
1
1
1
0
0
0
0
0
259.22
273.27
304.24
293.68
289.27
301.25
315.28
346.25
335.68
331.28
2.44
2.89
2.40
3.12
2.50
1.95
2.40
1.91
2.30
2.01
0
0
0
0
0
0
0
1
0
0
100.43
100.43
146.25
100.43
109.66
100.87
100.87
146.70
100.87
110.11
74.35 −4.39
74.35 −4.73
50.46 −4.85
74.35 −5.12
71.17 −4.41
74.20 −3.89
74.20 −4.23
50.61 −4.35
74.20 −4.62
71.01 −3.90
3.21
1.76
0.75
0.66
0.21
0.64
0.74
0.50
0.37
0.30
0.52
0.34
10
7
−4.04
4.28
8
2.96
8
0.02
8
−1.56
−5.36
1.45
11
8
9
−2.77
The calculation results show that all compounds meet the Lipinski rules of the five, suggesting that
these compounds theoretically would not have problems with oral bioavailability. Generally, all
compounds had scores of less than 5 for lipophilicity, ranging from 2.40 to 3.12 for the compounds
4a–e, and from 1.91 to 2.40 for 5a–e group. Most of the compounds also showed a PSA of less than
140 Ų, (indicating a good permeability of the drug in the cellular plasma membrane), this, with the
exception of compounds 4c and 5c, which showed higher values, The percentage of absorption (% ABS)
calculated ranged from 50.46 to 74.35 for the 4a–e group, and 50.61 to 74.20% for the 5a–e group,
see Table 1.
Most commercial products have LogS higher than −4.00 [30]. In Table 1, for the N-acylhydrazone
compounds 4a–e, LogS showed to be less than −4.00, ranging between −4.39 and −5.12, whereas for
the 1,3,4-oxadiazole compounds 5a–e showed LogS values in the range between −3.84 to −4.62.
Positive values for these drug scores indicate that the molecules contain predominantly
pharmacophoric groups, which are often found in pharmaceuticals. A positive value for drug-likeness
indicates that the compound contains predominantly fragments that are often present in most currently
used drugs [38]. The drug score combines drug-likeness, lipophilicity, solubility, molecular weight and
the risk of toxicity into a single numerical value that can be used to predict a global value for each
compound as a potential new drug candidate [38].
The results in the calculations show that the compounds gave values for drug-likeness between
−4.28 to 3.21, and −5.36 to 1.45 for 4a–e and 5a–e, respectively. All compounds showed positive
values in the drug score calculation, the values ranged from 0.21 to 0.75, and from 0.3 to 0.52 for the

Molecules 2012, 17
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compounds 4a–e and 5a–e respectively, see Table 1. The results show that these compounds,
especially compounds 4a and 5d, have potential as new drug candidates.
2.3. Antibacterial Activity
The N-acylhydrazones 4a–e [33] and the novel 1,3,4-oxadiazole compounds 5a–e show effective
antibacterial activity (MIC < 64 µg/mL [40]) for the various strains of S. aureus tested, with the MIC
values between 4 μg/mL and 32 μg/mL Table 2. The ratios of MBC/MIC were 1 or 2, indicating that
the anti-S. aureus effect of the compounds were bactericidal in nature (and not bacteriostatic) [41]
(Table 2).
Table 2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration
(MBC) of N-acylhydrazone compounds 4a–e and 1,3,4-oxadiazoles 5a–e against strains of
S. aureus.
Strains
Compound
SA-1199B
16-16 *
16-32
32-32
16-16
16-16
32-32
16-32
32-32
16-32
32-32
64-64
RN-4220
8-8
IS-58
8-8
007
8-8
05H
8-8
4a
4b
8-8
8-8
8-8
4-4
4c
16-32
8-8
16-16
8-16
8-16
8-8
8-8
4d
4-4
4e
16-16
16-32
16-32
16-32
16-32
32-32
64-128
8-8
8-8
4-4
5a
16-16
16-16
16-16
16-16
32-32
64-128
32-32
8-8
16-16
8-8
5b
5c
16-16
16-16
16-16
64-128
8-8
5d
5e
8-8
8-8
Chloramphenicol
64-128
* MIC-MBC (µg/mL).
In general, for all strains tested, the majority of 1,3,4-oxadiazole compounds 5a–e were less active
than their precursors 4a–e except for some compounds. For example, the compounds 5b–d showed
activity equal to the compounds 4b–d for the strain (SA-1199B), while the compound 5c was
equipotent to the compound 4c for the strains RN-4220, IS-58 and 05H. These results suggest that the
introduction of the nucleus 1,3,4-oxadiazole between the two aromatic rings in the derivatives 4a–e
was not as effective in obtaining of compounds more active than the original N-acylhydrazones.
Regarding the effluxing strains (SA-1199B, RN-4220, IS-58), the highest activity was exhibited by
compounds 4a, 4b, 4d, 5b and 5d. Regarding the MARSA strains (007 and 05H) the most active
compounds were 4b, 4d, 4e and 5b.
The biological activity exhibited by the compounds 4a–e and 5a–e shows a certain relationship with
the lipophilicity of the molecules. Indeed, it may be noted that in most cases there was a decrease in
activity of the compounds when the lipophilicity decreases. For example, when passing from the series
4a–e to the series 5a–e there is a reduction in lipophilicity of the compounds (Table 1), with a
concurrent decrease in biological activity (Table 2). Indeed, considering all strains tested, the more
lipophilic compounds 4b and 4d showed the best results of anti-staphylococcal activity.

Molecules 2012, 17
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Coincidentally, the drug chloramphenicol chosen as standard in the studies of anti-staphylococcal
activity has some structural similarity to the compounds 4a–e and 5a–e. In fact, this similarity is
clearer considering the structures of the compounds 4d, 5d and chloramphenicol and taking into
account three distinct regions in their structures: the region (A) containing the groups 4-chlorophenyl
in the compounds 4d and 5d and dichloromethylene in the structure of the chloramphenicol, the region
(B) containing the groups 4-nitrofuranyl in the compounds 4d and 5d and the 4-nitrophenyl in the
structure in the chloramphenicol and the region (C) represented in blue, containing both amide
functions (Figure 2).
Figure 2. Structural similarity of chloramphenicol with the compounds 4d and 5d.
O
B
B
C
HO
A
C
C
A
B
NO2
A
N
N
O
O
NO2
N
Cl
O
O
N
N
H
Cl
O
H
Cl
OH
NO2
Cl
(4d)
Chloramphenicol
(5d)
miLogP = 0.73
MIC = 64 g/mL
miLogP = 2.30
MIC = 8 g/mL
miLogP = 3.12
MIC = 4 g/mL
Interestingly, all compounds were more active than chloramphenicol, and for strain MARSA (05H)
the compound 4d was 16-fold more potent than the standard. This difference in activity is directly
related to the lipophilicity for compounds 4d, 5d and chloramphenicol (Figure 2). The increase in
polarity in the region (A) appears to contribute in a significant way to reduce the biological activity, as
shown for compounds 4d and 4c. Thus, in the structures of the compounds 4a–e and 5a–e, each of
which have the same basic skeleton, the difference in activity of these compounds is related to the
donor groups and electron withdrawing linked in the para position of benzene ring, thus providing
different lipophilicity to these compounds.
However, because of these promising results, an extension of the series of the compounds analogues
4a–e and 5a–e is under development in order to carry out quantitative structure-activity relationship
studies and thus have a better understanding on the relationship between the physicochemical
properties and biological activity observed for these compounds.
3. Experimental
3.1. Chemistry
All used reagents and solvents were purchased from commercial sources (Sigma-Aldrich, Brazil)
and used without a further purification. The progress of the reactions was monitored by thin layer
chromatography (TLC) on silica gel plates. The purification of the compounds was performed by
re-crystallization in ethanol and confirmed by determining the melting range on a MQAPF-3 brand
hotplate, and by means of gas chromatography with low resolution mass spectrometry (GCMS-QP2010)
Shimadzu. The spectra (IR) were obtained on a Shimadzu model IRPrestige-21 FTIR spectrometer,
13
using KBr pellets. 1H-NMR and C-NMR spectra were obtained on two different machines: a Varian
200 NMR (200 MHz for 1H and 50 MHz for 13C) and a Varian 500 NMR (500 MHz and 125 MHz for

Molecules 2012, 17
5102
1H and 13C, respectively), Deuterated dimethyl sulfoxide (DMSO-d6) was used as solvent, and
tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) were measured in units of parts
per million (ppm) and coupling constants (J) in Hertz (Hz).
3.2. General Procedure for the Preparation of N-Acylhydrazones of 4a–e
A mixture of aroylhydrazides 3a–e (3.0 mmol), 5-nitro-2-furaldehyde (0.423 g 3.0 mmol), absolute
ethanol (30.0 mL) and glacial acetic acid (6 drops) was heated under reflux for 3 h. Then the reaction
mixture was cooled to room temperature and poured into ice water. The precipitate formed was
filtered, washed with water and ethanol, and purified by ethanol recrystallization technique.
1
N'-((5-nitrofuran-2-yl)methylene)benzohydrazide (4a). H-NMR (200 MHz, δ ppm, DMSO-d6): 7.23
(d, 1H, J = 3.84, Hz, H-3), 7.76 (d, 1H, J = 3.84 Hz, H-4), 8.0–7.48 (m, 5H, ArH), 8.38 (s, 1H, H-6),
12.22 (s, br, 1H, NH).
4-Methyl-N'-((5-nitrofuran-2-yl)methylene)benzohydrazide (4b). 1H-NMR (200 MHz, δ ppm, DMSO-d6):
2.37 (s, 3H, CH3).7.90–7.20 (m, 6H, aromatic and furan), 8.39 (s, 1H, H-6), 12.15 (s, br, 1H, NH).
1
4-Nitro-N'-((5-nitrofuran-2-yl)methylene)benzohydrazide (4c). H-NMR (200 MHz, δ ppm, DMSO-d6):
7.30 (d, 1H, J = 3.6, Hz, H-3), 7.79 (d, 1H, J = 3.6 Hz, H-4), 8.14 (d, 2H, J = 9.0 Hz, H-12,14), 8.41
(d, 2H, J = 9.0 Hz, H-11,15), 8.41 (s, 1H, H-6), 12.48 (s, br, 1H, NH).
4-Chloro-N'-((5-nitrofuran-2-yl)methylene)benzohydrazide (4d). 1H-NMR (200 MHz, δ ppm, DMSO-d6):
7.26 (d, 1H, J = 3.96, Hz, H-3), 7.59 (d, 2H, J = 8.58 Hz, H-12,14), 7.78 (d, 1H, J = 3.9 Hz, H-4), 7.95
(d, 2H, J = 8.64 Hz, H-11,15), 8.38 (s, 1H, H-6), 12.26 (s, br, 1H, NH).
4-Methoxy-N'-((5-nitrofuran-2-yl)methylene)benzohydrazide (4e) 1H-NMR (200 MHz, δ ppm, DMSO-d6):
3.83 (s, 3H, OCH3), 7.06 (d, 2H, J = 9.48 Hz, H-12,14), 7.23 (d, 1H, J = 3.90, Hz, H-3), 7.77 (d, 1H,
J = 3.9 Hz, H-4), 7.91 (d, 2H, J = 8.82 Hz, H-11,15), 8.38 (s, 1H, H-6), 12.12 (s, br, 1H, NH).
3.3. General Procedure for the Preparation of 1-(2-(5-Nitrofuran-2-yl)-5-(4-substituted-phenyl)-1,3,4-
oxadiazol-3(2H)-yl)ethanones 5a–e
A mixture of N-acylhydrazones 4a–e (2.0 mmol) and acetic anhydride in excess (6.0 to 15.0 mL)
was heated under reflux at a temperature of 140 °C for 1 to 2.5 h. The reaction mixture was then
cooled to a temperature of 80–100 °C and poured into ice water (30.0 mL) to decompose the excess
acetic anhydride. The reaction mixture was stirred vigorously until a precipitate formed, which was
filtered, washed twice with aqueous NaHCO3 (5.0%), and then with water. The purification of the
compounds was performed by recrystallization from ethanol, or ethanol/water.
1-(2-(5-Nitrofuran-2-yl)-5-phenyl-1,3,4-oxadiazol-3(2H)-yl)ethanone (5a). Yield: 66.4%; m.p.
1
178–180 °C; H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.28 (s, 3H, H-14), 7.21 (d, 1H, J = 3.5Hz),
7.37 (s, 1H, H-8), 7.54 (t, 2H, J = 7.5 Hz), 7.61 (t, 1H, J = 7.5 Hz, H-1), 7.69 (d, 1H, J = 3.5 Hz,
H-11), 7.84 (d, 2H, J = 7.5 Hz, H-3,5); 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 21.04 (C-14), 84.34
(C-8), 113.2 (C-10), 114.54 (C-11), 123.31 (C-4), 126.57 (C-3,5), 129.12 (C-2,6), 132.15 (C-1), (C-9, no),

Molecules 2012, 17
5103
150.46 (C-12), 154.51 (C-7), 167.18 (C-13); IR (KBr): 1,670 (C=O), 1,631 (C=N), 1,242 (C-O-C),
1,535 and 1,361 (NO2), 1,593 and 1,500 (C=C) cm−1.
1-(2-(5-Nitrofuran-2-yl)-5-(methylphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5b). Yield: 45.9%; m.p.
160–162 °C; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.28 (s, 3H, H-14), 2.38 (s, 3H, CH3), 7.85–7.18
13
(m, 7H); C-NMR (125 MHz, DMSO-d6) δ (ppm): 21.53 (CH3), 21.56 (C-14), 84.58 (C-8), 113.61
(C-10), 114.80 (C-11), 120.93 (C-4), 126.10 (C-3,5), 130.43 (C-2,6), 142.84 (C-1), 150.47 (C-9), 150.95
(C-12), 155.04 (C-7), 167.49 (C-13); IR (KBr): 1,674 (C=O), 1,635 (C=N), 1,238 (C-O-C), 1,535 and
1,354 (NO2), 1,593 and 1,508 (C=C) cm−1.
1-(2-(5-Nitrofuran-2-yl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5c). Yield: 50.6%; m.p.
188–190 °C; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.31 (s, 3H, H-14), 7.26 (d, 1H, J = 3.5 Hz), 7.43
(s, 1H, H-8), 7.70 (d, 1H, J = 4.0 Hz, H-11), 8.06 (d, 2H, J = 8.5 Hz, H-3,5), 8.34 (d, 2H, J = 9.0 Hz,
13
H-2,6); C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.98 (C-14), 85.09 (C-8), 113.02 (C-10), 114.66
(C-11), 124.30 (C-2,6), 127.78 (C-3,5), 129.09 (C-4), 149.1 (C-1), 150.00 (C-12), 151.72 (C-9),
152.94 (C-7), 167.39 (C-13); IR (KBr): 1,670 (C=O), 1,627 (C=N), 1,246 (C-O-C), 1,543 and 1,354
(NO2), 1,597 and 1,516 (C=C) cm−1.
1-(2-(5-Nitrofuran-2-yl)-5-(chlorophenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5d). Yield: 74.3%);
m.p. 188–190 °C; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 2.28 (s, 3H, CH3), 7.23 (d, 1H, J = 4.0 Hz,
H-10), 7.39 (s, 1H, H-8), 7.61 (d, 2H, J = 9.00 Hz, H-2,6), 7.70 (d, 1H, J = 4.0 Hz, H-11), 7.84 (d, 2H,
J = 8.50 Hz, H-3,5); 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 20.98 (C-14), 84.59 (C-8), 113.07 (C-10),
114.47 (C-11), 122.17 (C-4), 128.30 (C-3,5), 129.27 (C-2,6), 136.79 (C-1), 150.26 (C-12), (C-9, no),
153.66 (C-7), 167.16 (C-13); IR (KBr): 1,670 (C=O), 1,631 (C=N), 1,242 (C-O-C), 1,535 and 1,357
(NO2), 1,593 and 1,500 (C=C) cm−1.
1-(2-(5-Nitrofuran-2-yl)-5-(methoxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5e). Yield: 40.8%;
1
m.p. 181–183 °C; H-NMR (200 MHz, DMSO-d6) δ (ppm): 2.27 (s, 3H, CH3), 3.84 (s, 3H, OCH3),
7.08 (d, 2H, J = 8.64 Hz, H-2,6), 7.20 (d, 1H, J = 3.90 Hz, H-10), 7.34 (s, 1H, H-8), 7.70 (d, 1H,
13
J = 3.54 Hz, H-11), 7.79 (d, 2H, J = 8.82 Hz, H-3,5); C-NMR (50 MHz, DMSO-d6) δ (ppm): 21.49
(C-14), 55.89 (OCH3), 84.42 (C-8), 113.61 (C-10), 114.82 (C-11), 114.97 (C-2,6), 115.81 (C-4),
128.93 (C-3,5), 151.03 (C-12), 152.16 (C-9), 154.94 (C-7), 162.59 (C-1), 167.37 (C-13); IR (KBr):
1,662 (C=O), 1,635 (C=N), 1,257 (C-O-C), 1,535 and 1,357 (NO2), 1,608 and 1,504 (C=C) cm−1.
3.4. Bacterial Strains
The strains of S. aureus used were: SA-1199B, which overexpresses the NorA gene encoding the
NorA efflux protein for fluoroquinolones (and other drugs) [42]; RN4220, harbouring plasmid
pUL5054, which carries the gene encoding the MsrA macrolide efflux protein [43]; IS-58, which
possesses the TetK tetracycline efflux protein [44], and two clinical strains (007 and 005H), resistant to
amino glycosides and to methicillin (MARSA). The effluxing strains were kindly provided by
Professor Simon Gibbons (University of London, London, UK), and the MARSA strains were
obtained from the culture collection of the Laboratory of Microorganism Genetics (Department of

Molecules 2012, 17
5104
Molecular Biology, Federal University of Paraíba). All strains were maintained on blood agar base
slants (BAB, Difco Laboratories Ltd., Brazil), and prior to use, the cells were grown overnight at 37 °C
in brain heart infusion broth (BHI, Difco Laboratories Ltd., Detroit, MI, USA).
3.5. Antibacterial Activity
The stock solutions of the compounds were prepared in DMSO which at its highest final
concentration after dilution in the broth (4%) caused no inhibition of bacterial growth. The minimum
inhibitory concentrations (MICs) of the compounds were determined in BHI by microdilution assay
using a suspension of ca. 105 cfu/mL and a drug concentration range of 256 to 0.5 μg/mL (two-fold
serial dilutions). The MIC is defined as the lowest concentration at which no growth is observed. The
dye resazurin was used for better visualization of the bacterial growth. Subcultures were made on BAB
from the last well that showed visible growth, and from all successive wells. The minimum
bactericidal concentration (MBC) is defined as the lowest drug concentration that gives no growth on
the agar.
4. Conclusions
We have described the synthesis and evaluation of anti-staphylococcal activity for
N-acylhydrazones 4a–e, and five new 1,3,4-oxadiazole compounds 5a–e against several strains of
1
Staphylococcus aureus. All compounds were fully characterized using IR, H- and 13C-NMR
spectroscopic techniques. In studies of anti-staphylococcal activity, all the compounds exhibited good
results with MIC between 4 μg/mL and 32 μg/mL, being more potent than the standard drug
chloramphenicol. In silico studies, all compounds had a desirable profile to be new drug candidates.
Acknowledgments
J.P.S-J. and V.S.F-S. are very grateful to Simon Gibbons (University of London), for his valuable
and kind cooperation. This work was supported by the following Brazilian agencies: CNPq, CAPES,
PRONEX/FAPESQ-PB.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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