Synthesis and testing of 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4- oxadiazole derivatives for antifungal activity against selected Candida species

Article abstract of DOI:10.1590/S0103-50532013000100016

A series of 21 1,3,4-oxadiazoline derivatives was synthesized by cyclization of N-acylhydrazones with acetic anhydride and evaluated for their in vitro antifungal activity against six Candida strains: Candida albicans (ATCC 90028 and LM V-42), C. krusei (ATCC 6258 and LM 12 C) and C. tropicalis (ATCC 13803 and LM 14). The Candida strains were found to be sensitive to some of the compounds, which inhibited the growth by 50-90percent, with minimum inhibitory concentration (MIC) in the range of 64-512 μg mL^-1. The compounds' structures were fully confirmed and characterized by Fourier transform infrared spectroscopy (FTIR), ¹H and ¹³C nuclear magnetic resonance (NMR) and mass spectrometry (MS).

Full text of DOI:10.1590/S0103-50532013000100016

J. Braz. Chem. Soc., Vol. 24, No. 1, 115-120, 2013.
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Article
A
Synthesis and Testing of 3-Acetyl-2,5-Disubstituted-2,3-Dihydro-1,3,4-oxadiazole
Derivatives for Antifungal Activity Against Selected Candida Species
Cledualdo S. de Oliveira,a Bruno F. Lira,a José M. Barbosa-Filho,b Jorge G. F. Lorenzo,b
Camilla P. de Menezes,b Jessyca M. C. G. dos Santos,b Edeltrudes de O. Limab and
Petrônio F. de Athayde-Filho*,a
aDepartamento de Química, Universidade Federal da Paraíba, 58051-900 João Pessoa-PB, Brazil
bDepartamento de Ciências Farmacêuticas, Universidade Federal da Paraíba,
58051-900 João Pessoa-PB, Brazil
Uma série de 21 derivados, 1,3,4-oxadiazolinas, foi sintetizada pela ciclização de
N-acilhidrazonas com anidrido acético e avaliada in vitro quanto à sua atividade antifúngica
contra seis espécies de Candida: Candida albicans (ATCC 90028 e LM V-42), Candida krusei
(ATCC 6258 e LM 12 C) e Candida tropicalis (ATCC 13803 e LM 14). As espécies de Candida
foram consideradas sensíveis a uma série dos compostos, os quais inibiram o crescimento de
50 a 90%, com um intervalo de concentração inibitória mínima (MIC) de 64 a 512 µg mL-1.
As estruturas dos compostos foram totalmente confirmadas e caracterizadas pelas técnicas de
infravermelho com transformada de Fourier (FTIR), ressonância magnética nuclear (NMR) de
1H e 13C, e espectrometria de massa (MS).
A series of 21 1,3,4-oxadiazoline derivatives was synthesized by cyclization of N-acylhydrazones
with acetic anhydride and evaluated for their in vitro antifungal activity against six Candida strains:
Candida albicans (ATCC 90028 and LM V-42), C. krusei (ATCC 6258 and LM 12 C) and C.
tropicalis (ATCC 13803 and LM 14). The Candida strains were found to be sensitive to some of
the compounds, which inhibited the growth by 50-90%, with minimum inhibitory concentration
(MIC) in the range of 64-512 µg mL-1. The compounds’ structures were fully confirmed and
1
characterized by Fourier transform infrared spectroscopy (FTIR), H and 13C nuclear magnetic
resonance (NMR) and mass spectrometry (MS).
Keywords: 1,3,4-oxadiazoline, antifungal activity, Candida species, N-acylhydrazones
Introduction
Therefore, taking into consideration our ongoing
research and the importance of the 1,3,4-oxadiazoline
scaffold as a good source for the discovery of new and
biologically active molecules, our group synthesized new
1,3,4-oxadiazoline heterocyclic compounds, (specifically
3-acetyl-2,5-diaryl-2,3-dihydro-1,3,4-oxadiazoles), and
pre-screened these compounds for candidacy as novel
antifungal agents.
Due to the development of resistant fungous strains and
no-longer effective medications, the incidence of systemic
fungal infections (with its consequent morbidity and
mortality) has been gradually increasing over the last three
decades.1 The resulting requirement for investigation,
research and discovery of new antimicrobial agents that are
both safer and more effective is of the utmost importance.
Derivatives of 1,3,4-oxadiazoline have recently gained
importance (for serving as building blocks) for both organic
synthesis and medicinal chemistry when developing new
therapeutic agents. It has been noted that compounds
containing the 1,3,4-oxadiazoline motif generally exhibit
excellent antimicrobial activity.2-12
Results and Discussion
Chemistry
N-acylhydrazones (NAH) belong to the azomethine
class of compounds, they are structurally privileged with
chemical properties that have proven to be important when
designing pharmacologically active prototypes.13,14 They are
*e-mail: athayde-filho@quimica.ufpb.br

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Synthesis and Testing of 3-Acetyl-2,5-Disubstituted-2,3-Dihydro-1,3,4-oxadiazole
J. Braz. Chem. Soc.
particularly versatile for synthesis of compounds containing
nitrogen.15 With this in mind, it was planned the synthesis
of the 1,3,4-oxadiazoline target molecule from the versatile
N-acylhydrazone substrate, using the strategy of closing
the acyclic chain to give the 1,3,4-oxadiazoline core. The
strategy is efficient to obtain the 1,3,4-oxadiazoline target
molecule (Figure 1).
linked to the aromatic ring at the 2-position of the core
1,3,4-oxadiazoline, series 2 (2-(4-acetoxyphenyl)-3-acetyl-
5-aryl-2,3-dihydro-1,3,4-oxadiazole, 4m-q), in which the
p-acetoxyphenyl group remained fixed at the 2-position of
the 1,3,4-oxadiazoline ring, and series 3 (2-(5-nitrofuran-
2-yl)-3-acetyl-5-aryl-2,3-dihydro-1,3,4-oxadiazole, 4r-v)
having the group 5-nitrofuran-2-yl fixed at the 2-position of
the 1,3,4-oxadiazoline ring. In both series 2 and 3, electron-
donating and electron-withdrawing groups are located at
the para position of the aromatic ring and at the 5-position
of the 1,3,4-oxadiazoline core, (Figure 2).
Compounds 4a, 4c-g and 4i are known structures.16,17
Compounds of series 2 (4m-q) and compounds 4b, 4h,
4j and 4l are new structures. Compounds of series 3 (4r-v)
were recently reported by our research group.2 The purity
of the compounds was verified examining their melting
ranges and by gas chromatography (GC-MS) (Table 1).
Interestingly, all the compounds contain an acetyl group
(which is polar) in the 3-position of the 1,3,4-oxadiazoline
core, which gives to these compounds an intermolecular
hydrogen bonding site with bio-macromolecules.
Figure 1. Retrosynthetic analysis of 1,3,4-oxadiazoline.
Target molecules 4a-v were obtained as racemic
mixtures in four synthetic steps according to Scheme 1.
The N-acylhydrazone compounds (3) were obtained from
a condensation reaction between acylhydrazides, and
aromatic/hetero-aromatic aldehydes according to the
literature.2 The cyclization reaction of the derivatives (3)
with acetic anhydride afforded 1,3,4-oxadiazoline
derivatives 4a-v in yields that were moderate to good
(45.4-81.2%).
Twenty-one 1,3,4-oxadiazoline compounds were
synthesized and divided into three congener sets as follows:
series 1 (2-aryl-3-acetyl-5-(pyridin-4-yl)-2,3-dihydro-
1,3,4-oxadiazole, 4a-l), containing the pyridin-4-yl group
fixed in 5-position of the 1,3,4-oxadiazoline ring, with
varied electron- donating and electron-withdrawing groups
Characterization of the final products
The structures of the compounds were fully
characterized and confirmed by using spectroscopic
techniques (Fourier transform infrared (FTIR)
spectroscospy and 1H and 13C nuclear magnetic resonance
(NMR)) and mass spectrometric (MS) studies. The 1H and
13C NMR spectroscopic techniques were sufficient to
confirm the formation of the 1,3,4-oxadiazoline ring
Scheme 1. Reagents and conditions: (i) EtOH, H2SO4 (Cat), reflux (4 h), (ii) EtOH, hydrazine hydrate, reflux (3 h), (iii) EtOH, aromatic aldehydes, AcOH
(cat.), reflux (3 h), and (iv) acetic anhydride, reflux (2 h).
Figure 2. The chemical structures of the 1,3,4-oxadiazoline compounds.

Vol. 24, No. 1, 2013
Oliveira et al.
117
Table 1. Physical and chemical properties of novel 1,3,4-oxadiazolines 4b, 4h, 4j, 4l and 4m-q
GC-MS
tr / min
23.4
Compound
Molecular formula
Exact mass
mp / °C
Yield / %
4b
4h
4j
C18H19N3O2
C15H12BrN3O2
C17H17N3O4
C17H15N3O4
C18H16N2O4
C19H18N2O4
C18H15N3O6
C18H15ClN2O4
C19H18N2O5
309.15
345.01
327.12
325.11
324.11
338.13
369.10
358.07
354.12
92-94
66
59
57
72
75
70
64
71
45
120-121
186-188
162-164
140-141
154-156
192-194
158-160
98-100
24.1
24.3
4l
24.5
4m
4n
4o
4p
4q
24.3
25.3
25.8
25.7
26.5
because of its very characteristic signals.2,17 For example,
in the 1H NMR spectra of compounds of the series 1 (4a-l),
two typical signals were observed, one assigned to the
methyl protons of the acetyl group linked to the nitrogen
atom N-3 in the aliphatic region of 2.27 to 2.30 ppm, and
another assigned to methinic protons H-2 in the aromatic
region 7.17 to 7.63 ppm. In the 13C NMR spectra, signals
characteristic of C=O around 167 ppm, and methyl carbon
atom CH3 in the range of 20.1-22.7 ppm, as well as the
oxadiazole ring signal C-2 around 88.7 to 92.9 ppm, and
C-5 around 152.5 to 163.4 ppm were observed, thus
confirming its formation.
C-O-C absorption stretches (oxadiazole ring) from 1095
to 1240 cm-1, and C=N absorptions (oxadiazole ring) from
1604 to 1635 cm-1. The compound 4-(3-acetyl-5-(pyridin-
4-yl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)phenyl acetate
4l and all compounds of series 2 (4m-q) also showed
absorption bands in the 1755 to 1759 cm-1 range for C=O
(acetoxy group).
The fragmentation most observed in the mass spectra of
the new compounds (4b, 4g, 4j, 4l and 4m-q) is exemplified
by compound 4l. In the mass spectrum of compound 4l,
it was not detected a peak related to the molecular ion
(m/z 325), this due to the presence of an acetyl radical
which easily promotes the loss of one ketene molecule, and
the transfer of a hydrogen to form a phenol. However, the
radical cation (m/z 283) originating from the first breaking
of the molecular ion, and corresponding to the elimination
of a neutral ketene, was not observed. The peak at m/z 241
originates from the radical cation (m/z 283) through loss
of a second neutral ketene molecule. The base peak was
assigned to the acylium ion (m/z 43). In Scheme 2, it is
outlined a fragmentation pattern proposal for that observed
in the mass spectrum of compound 4l.
In the 1-(2-(4-fluorophenyl)-5-(pyridin-4-yl)-
1,3,4-oxadiazol-3(2H)-yl)ethanone, (4f) spectrum, it was
observed doublets corresponding to couplings 13C-19F, whose
coupling constants for one bond 1JCF 245.0 Hz, two bonds
3
2JCF 32.0 Hz and three bonds JCF 9.0 Hz were assigned
respectively to couplings C-F in d 162.4 (C-14), d 115.4
1
(C-11,15) and d 128.8 (C-12,16) ppm. In the H NMR
spectra for series 2 (4m-q) compounds, it was observed
the characteristic signals: a singlet for hydrogen 3 (acetyl
group linked to nitrogen (N-3)) in the aliphatic region of
2.25 to 2.27 ppm, a singlet for hydrogen 3 (acetoxy group
linked to the benzene ring) in the aliphatic region of (2.26 to
2.30 ppm), and singlet for hydrogen 1 (H-2) in the aromatic
Antifungal activity
13
region of 7.17 to 7.26 ppm. In the C NMR spectra, the
The in vitro antifungal activity of compounds 4a-v was
evaluated by the microdilution method against six strains
of pathogenic fungi, Candida albicans (ATCC 90028 and
LMV-42), Candida krusei (ATCC 6258 and LM 12 C) and
Candida tropicalis (ATCC 13803 and LM 14), using
nystatin as the drug standard (Table 2). The compounds
were tested at concentrations from 32 to 1024 µg mL-1, and
solubilized in dimethylsulfoxide, DMSO (Sigma Chemical)
to 10% to avoid interferences with the microorganisms.
The antifungal activity of the products was interpreted, and
considered active or not, according to the following
characteristic signals observed were: two carbon atoms of
the 1,3,4-oxadiazoline core (C-2 and C-5) in the regions
of 91.0 to 92.3 ppm and 153.2 to 154.7 ppm, respectively.
The chemical shifts of C=O of the acetyl group linked to
nitrogen atom (N-3), and the C=O of the acetoxy group
were observed in the range of 166.6 to 167.2 ppm and 168.8
to 169.2 ppm, respectively. The characterization of series
3 (4 r-v) compounds was recently reported by our group.2
In the infrared spectrum, compounds 4a-q showed
amide C=O absorption bands from 1662 to 1674 cm-1,

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Synthesis and Testing of 3-Acetyl-2,5-Disubstituted-2,3-Dihydro-1,3,4-oxadiazole
J. Braz. Chem. Soc.
Scheme 2. Fragmentation pattern proposal for the compound 4l.
Table 2. Antifungal activity of 1,3,4-oxadiazoline 4m-q and 4r-v
Minimum inhibitory concentration (MIC) / (µg mL-1)
Candida Strains
Compounds
C. albicans
C. albicans
C. krusei
C. krusei
C. topicalis
C. topicalis
ATCC 90028
LM V-42
ATCC 6258
LM 12C
ATCC 13803
LM 14
4m
R
R
1024
128
R
R
128
R
1024
R
R
R
R
R
4n
4o
R
R
R
R
4p
R
R
R
R
R
R
4q
R
R
R
R
R
R
4r
64
128
128
512
64
+
64
128
R
256
128
256
128
64
+
R
512
R
512
128
256
64
128
+
4s
R
4t
R
R
4u
128
128
+
R
R
4v
R
128
+
Control yeast
Nystatin
+
-
-
-
+
+
-
R: resistant; +: growth of the microorganism; -: no growth of the microorganism.
parameters: 50-100 µg mL-1 = good activity; 100-
500 µg mL-1 = moderate activity; 500-1000 µg mL-1 = low
activity; greater than 1000 µg mL-1 = inactive product.18
From the 21 compounds tested, compounds 4a-m and
4o-q showed no inhibitory activity against the mentioned
yeasts in the bioassays. Compound 4n showed moderate
inhibitory effect against Candida albicans (LMV-42), and
Candida krusei (ATCC 6258) at a minimum concentration
of 128 µg mL-1. The Candida species were sensitive to
compounds of series 3 (4r-v), these compounds inhibited
the growth by 50 to 90% (MIC range of 64-512 µg mL-1). The
best results were against Candida albicans (ATCC 90028)
exhibited by compounds 4r and 4v with an MIC of 64 µg
mL-1, while compounds 4u and 4v exhibited the best results
against Candida krusei, and Candida tropicalis with an
MIC of 64 µg mL-1, respectively. Candida krusei (LM 12C)
showed no sensitivity to any of the tested compounds. The
standard drug, nystatin at 100 IU mL-1 inhibited the growth
of four (67%) of the Candida strains. The results therefore
were considered between good and moderate in terms of
biological activity, and taking into account the parameters
established.18 The results show that compounds 4r-v may
be considered promising for possible development of new
antifungal agents, and, that the 5-nitrofuranyl group in these
compounds is important for their activity.
Conclusions
In this work, it was synthesized a series of twenty-one
1,3,4-oxadiazoline compounds, and their structures were
confirmed by the techniques FTIR 1H and 13C NMR and
MS. For in vitro antifungal activity against Candida
strains, compounds 4r-v showed good to moderate
activity, inhibiting the growth of the strains used by

Vol. 24, No. 1, 2013
Oliveira et al.
119
50-90% with an MIC of 64-512 µg mL-1. These may
be promising compounds for the development of new
antifungal agents.
Determination of minimum inhibitory concentration (MIC)
The MIC value was determined by the microdilution
method, using 96 well microtiter plates with background in a
“U” and in duplicate. To each well of the plate, it was added
100 µL of liquid medium SDB doubly concentrated. Then,
100 µL of the product solution (also doubly concentrated)
were dispensed into the wells of the first line of the plate.
By means of serial dilution (ratio of two), it was obtained
concentrations from 2,048 to 64 µg mL-1, so that in the
first row of the plate, the highest concentration, and in the
latter, the lower concentrations. Finally, 10 µL of inoculum
were added to the wells where each column of the plate
referred specifically to a strain. The same was also done
in the culture medium with fungal drug nystatin (100 UI).
The plates were incubated at 37 °C for 24-48 h. For each
strain, MIC was defined as the lowest concentration able
to inhibit fungal growth visually observed in the wells,
when compared to the control. All tests were performed in
duplicate, and the results were expressed as the geometric
mean of the MIC values obtained in the two trials.21-23
Experimental
Chemistry
All used reagents and solvents were purchased
from commercial sources (Sigma-Aldrich, Brazil), and
used without a further purification. The purification of
the compounds was performed by re-crystallization in
ethanol, and confirmed by determining the melting range
on an MQAPF-3 hotplate, and by gas chromatography
with low resolution mass spectrometer (GC-MS-QP2010)
Shimadzu. The FTIR spectra were obtained on an FTIR
spectrometer model IRPrestige-21-Shimadzu, using KBr
1
pellets. H and 13C NMR spectra were obtained on two
different machines: a Varian 200 NMR (200 and 50 MHz
for 1H and for 13C, respectively), and a Varian 500 NMR
1
(500 and 125 MHz for H and 13C, respectively),
deuterated dimethyl sulphoxide (DMSO-d6) was used
as the solvent and tetramethylsilane (TMS) was used for
the internal standard. Chemical shifts (d) were measured
in parts per million (ppm) and the coupling constants
(J) in Hz.
Acknowledgments
This work was supported by the following Brazilian
agencies: Conselho Nacional de Desenvolvimento Científico
eTecnológico (CNPq) and Coordenação deAperfeiçoamento
de Pessoal de Nível Superior (CAPES). The English in this
article was reviewed by David Peter Harding.
Antifungal activity
Microorganisms
Supplementary Information
The microbiological assays used: Candida albicans
(ATCC 90028 and LM V-42), C. krusei (ATCC 6258 and
LM 12 C) and C. tropicalis (ATCC 13803 and LM 14).
The strains were acquired from the InstituteAdolfo Lutz of
São Paulo and Mycology Laboratories of the Departments
of Pharmaceutical Sciences at the University of Sao
Paulo and the Federal University of Paraiba, respectively.
The fungal strains were maintained in appropriate medium,
Sabouraud Dextrose Broth-SDB (DIFCO Laboratories,
France-USA), and stored at 4 °C and 35 °C. The
suspension of microorganisms was prepared according
to the 0.5 McFarland scale tube, and adjusted by means
of a spectrophotometer (Leitz-Photometer 340-800)
to 90% T (530 nm) corresponding to approximately
106 UFC mL-1.19-21
Supplementary information is available free of charge
at http://jbcs.sbq.org.br as PDF file.
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