Synthesis and antibacterial evaluation of 3,5-Diaryl-1,2,4-oxadiazole derivatives

Article abstract of DOI:10.21577/0103-5053.20180118

This manuscript reports the synthesis of twenty 3,5-diaryl-1,2,4-oxadiazole derivatives, nine of which are novel compounds. The amidoxime reaction with acyl chlorides obtained from substituted benzoic acids was used. All compounds were tested against five standard (American Type Culture Collection (ATCC)) bacteria: Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis and Staphylococcus aureus. Screening assays were carried out using agar-diffusion technique, in which 100 μM heterocyclic compounds solutions (20percent dimethylsulfoxide/water) were employed. The minimum inhibitory concentrations (MIC) of the active compounds were determined by serial dilutions at decreasing concentrations in microtiter plates. The nitrated derivatives gave the best test results, where MIC = 60 μM (E. coli) was the lowest value found for an ortho-nitrated derivative. The activity of these compounds possibly involves a mechanism via free radicals. S. aureus and P. aeruginosa were resistant to all compounds.

Full text of DOI:10.21577/0103-5053.20180118

http://dx.doi.org/10.21577/0103-5053.20180118
J. Braz. Chem. Soc., Vol. 29, No. 11, 2405-2416, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Synthesis and Antibacterial Evaluation of 3,5-Diaryl-1,2,4-oxadiazole Derivatives
Felipe S. Cunha,a Joseli M. R. Nogueirab and Alcino P. de Aguiar*,a
aLaboratório de Síntese Orgânica, Seção de Engenharia Química, Instituto Militar de Engenharia,
Praça General Tibúrcio, 80, Praia Vermelha, Urca, 22290-270 Rio de Janeiro-RJ, Brazil
bEscola Nacional de Saúde Pública (ENSP), Fundação Oswaldo Cruz (Fiocruz),
Rua Leopoldo Bulhões, 1480, Manguinhos, 21041-210 Rio de Janeiro-RJ, Brazil
This manuscript reports the synthesis of twenty 3,5-diaryl-1,2,4-oxadiazole derivatives, nine of
which are novel compounds. The amidoxime reaction with acyl chlorides obtained from substituted
benzoic acids was used. All compounds were tested against five standard (American Type Culture
Collection (ATCC)) bacteria: Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis,
Proteus mirabilis and Staphylococcus aureus. Screening assays were carried out using agar-
diffusion technique, in which 100 μM heterocyclic compounds solutions (20% dimethylsulfoxide/
water) were employed. The minimum inhibitory concentrations (MIC) of the active compounds
were determined by serial dilutions at decreasing concentrations in microtiter plates. The nitrated
derivatives gave the best test results, where MIC = 60 μM (E. coli) was the lowest value found for
an ortho-nitrated derivative. The activity of these compounds possibly involves a mechanism via
free radicals. S. aureus and P. aeruginosa were resistant to all compounds.
Keywords: oxadiazole, amidoxime, cyclization, nitrocompound, antibacterial
Introduction
antibacterial properties, as well as simplified synthetic
routes. The oxadiazole nucleus presents four isomers
(Figure 1); however, one of them, the 1,2,3-oxadiazole
isomer, is not of any practical use, as it is unstable and
decomposes easily.4
Although the 1,2,4-oxadiazole heterocycle was first
synthesized in 1884,5 it was only in the 1990s that this
nucleus aroused interest within the scientific community.6
The literature reports a diverse set of applications for
the 1,2,4-oxadiazole core in medicinal chemistry,7,8
such as antiasthmatic,9 antidiabetic,10 antitumoral,11
anti-inflammatory,12 antioxidant,12 and antimicrobial.13
Different parameters are used to quantify these biological
activities, such as half maximal inhibitory concentration
(IC50), half maximal effective concentration (EC50) or
minimum inhibition concentration (MIC) (Figure 2). In
Materials Science, structures containing this heterocycle
The increasing bacterial resistance to known antibiotics
has been causing concern in the population, since their
therapeutic possibilities have been diminishing.1 This has
led to intensified research involving the synthesis and
evaluation of heterocyclic derivatives with antimicrobial
properties, such as 1,2,4-oxadiazoles.2
The indiscriminate use of antibiotics is pointed
out as one of the main causes for the development of
bacterial resistance. The growth of bacterial resistance has
caused the mortality of an expressive number of people
worldwide. Even the main commercial antimicrobial
compounds (e.g., ampicillin, posaconazole, ciprofloxacin)
may have limited action, nowadays, against strains
of resistant microorganisms.3 Therefore, there is an
urgent need to find new molecules with more efficient
Figure 1. Isomers of oxadiazoles.
*e-mail: alcino@ime.eb.br

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Synthesis and Antibacterial Evaluation of 3,5-Diaryl-1,2,4-oxadiazole Derivatives
J. Braz. Chem. Soc.
Figure 2. Examples of bioactive 1,2,4-oxadiazoles.
have technological applications in ionic liquids and liquid
crystals, for example.14-16
constant (J) is given in hertz (Hz). Multiplicities were
presented as singlet (s), doublet (d), triplet (t), quadruplet
(q) and multiplet (m). Melting points were determined in
open capillary tubes (Gehaka PF 1000, Brazil) and are
uncorrected.A gas chromatograph,Agilent 7890A (USA),
coupled to a nitrogen and phosphorus detector (NPD) and a
mass spectrometer,Agilent MSD 5975C (USA) were used.
The 1,2,4-oxadiazole derivatives can be synthesized via
two classical methods: 1,3-dipolar cycloaddition of nitrile
oxides to the nitrile functional group17 and the cyclization of
amidoxime derivatives,18 the latter being the most common.
This paper reports the synthesis of 3,5-diphenyl-
1,2,4-oxadiazoles and 3-pyridino-5-phenyl-
1,2,4-oxadiazole derivatives employing the amidoxime
route. These products have different substitution patterns
(position or nature of the substituents).All compounds had
their antibacterial properties evaluated against five bacteria
(Gram-positive and Gram-negative).
Synthesis of amidoximes (1-4)
Solutions of 50 mmol of hydroxylamine hydrochloride,
25 mmol of anhydrous sodium carbonate and 20 mL of
water were added to a round-bottomed flask. The mixture
was homogenized until no effervescence occurred. Then,
33 mmol of a nitrile derivative (1-4) and 20 mL of ethanol
were added to the reaction medium, which was kept
under stirring and refluxed for 5 h. After this period, the
product, an amidoxime (5-8), was recrystallized from water
(Scheme 1).19
Experimental
Materials and methods
All reagents and solvents were purchased (Aldrich,
Brazil) and used without further purification.All reactions,
except the synthesis of the amidoximes, were performed
in a closed system to protect against moisture. The
reactions were monitored by thin layer chromatography
(TLC) using pre-coated silica gel 60 F254 plates (Merck,
Brazil). Visualization of the plates was accomplished
by using UV light. Infrared spectra (IR) were obtained
on a PerkinElmer Spectrum 100 (USA) spectrometer.
1H and 13C nuclear magnetic resonance (NMR) spectra
were obtained on a Bruker AVANCE DPX 400 (USA)
spectrometer. Chemical shifts (d) are reported in ppm
relative to tetramethylsilane. The solvent used was CDCl3
and dimethylsulfoxide-d6 (DMSO-d6) and the coupling
Synthesis of acyl chloride (9-19)
Thionyl chloride (5 mL) was slowly added to a round-
bottomed flask containing carboxylic acid (3.1 mmol). The
medium was refluxed for 2 h. Only acyl chloride (9-19)
remained in the flask after the excess thionyl chloride was
removed by distillation.20
Synthesis of the 1,2,4-oxadiazol derivatives (20-39)
The newly synthesized acyl chloride dissolved in
toluene (10 mL) was slowly added to a solution of

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Cunha et al.
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Scheme 1. Synthetic route of the 1,2,4-oxadiazol derivatives.
amidoxime (2.5 mmol) dissolved in toluene (40 mL) in a
250 mL round-bottomed flask at 25 oC and under constant
stirring. After the addition, the solution was refluxed for
20 h, and then washed with sodium carbonate solution. The
organic phase was dried with anhydrous sodium sulfate
and thereafter the toluene was removed under vacuum
by heating. A solid was obtained, which was purified by
flash column chromatography using a hexane/ethyl acetate
mixture as eluent with an increasing polarity gradient (9:1
to 6:4).20
3-(4-Chlorophenyl)-5-(2-chlorophenyl)-1,2,4-oxadiazole
(22)
o
1
Yield: 49%; mp 128-130 C; H NMR (400.00 MHz,
CDCl3) d 8.15 (dd, 1H, J 6.0, 1.6 Hz), 8.12 (d, 2H,
J 8.0 Hz), 7.60 (dd, 1H, J 8.0, 1.2 Hz), 7.52 (td, 1H, J 7.6,
1.2 Hz), 7.49 (d, 2H, J 8.0 Hz), 7.44 (td, 1H, J 7.6, 1.2 Hz);
13C NMR (100.0 MHz, CDCl3) d 174.5, 167.9, 137.4, 133.9,
133.2, 131.9, 131.5, 129.2, 128.9, 127.1, 125.3, 123.5;
IR (KBr) n / cm-1 1597, 1543, 1411, 1357, 1087, 833,
740; HRMS (ESI) m/z, calcd. for C14H8Cl2N2O [M + H]+:
291.0086, found: 291.0082.
3-Phenyl-5-phenyl-1,2,4-oxadiazole (20)
o
1
Yield: 76%; mp 108-110 C; H NMR (600.00 MHz,
CDCl3) d 8.80 (d, 2H, J 7.8 Hz), 8.76 (d, 2H, J 6.0 Hz),
8.18 (t, 1H, J 7.8 Hz), 8.13 (t, 2H, J 7.8 Hz), 8.10 (d, 3H,
J 6.0 Hz); 13C NMR (150.0 MHz, CDCl3) d 175.7, 168.9,
132.7, 131.2, 129.1, 128.8, 128.1, 127.5, 126.9, 124.3; IR
(KBr) n / cm-1 1612, 1600-1390, 735, 695; high resolution
mass spectrometry (HRMS) (electrospray ionization (ESI))
m/z, calcd. for C14H10N2O [M + H]+: 223.0865, found:
223.0867.
3-(4-Chlorophenyl)-5-(2-bromophenyl)-1,2,4-oxadiazole
(23)
o
1
Yield: 45%; mp 130-134 C; H NMR (400.00 MHz,
CDCl3) d 8.14 (d, 2H, J 8.8 Hz), 8.08 (dd, 1H, J 7.6, 2.0 Hz),
7.80 (dd, 1H, J 7.6, 1.2 Hz), 7.50 (t, 1H, J 6.4 Hz), 7.48
(d, 2H, J 8.4 Hz), 7.44 (td, 1H, J 7.6, 2.0 Hz); 13C NMR
(100.0 MHz, CDCl3) d 175.0, 167.9, 137.5, 134.9, 133.2,
132.1, 129.2, 128.9, 127.6, 125.6, 125.3, 122.2; IR (KBr)
n / cm-1 1589, 1535, 1411, 910, 833, 740; HRMS (ESI)
m/z, calcd. for C14H8BrClN2O [M + H]+: 334.9576, found:
334.9581.
3-(4-Chlorophenyl)-5-phenyl-1,2,4-oxadiazole (21)
o
1
Yield: 68%; mp 111-113 C; H NMR (400.00 MHz,
CDCl3) d 8.20 (d, 2H, J 8.0 Hz), 8.11 (d, 2H, J 8.0 Hz),
7.61 (t, 1H, J 8.0 Hz), 7.55 (t, 2H, J 8.0 Hz), 7.48 (d, 2H,
J 8.0 Hz); 13C NMR (100.0 MHz, CDCl3) d 175.9, 168.2,
137.3, 132.9, 129.2, 129.1, 128.8, 128.2, 125.5, 124.1; IR
(KBr) n / cm-1 1604, 1550, 1489, 1404, 1037, 840, 740,
695; HRMS (ESI) m/z, calcd. for C14H9ClN2O [M + H]+:
257.0476, found: 257.0480.
3-(4-Chlorophenyl)-5-(2-iodophenyl)-1,2,4-oxadiazole (24)
o
1
Yield: 48%; mp 91-93 C; H NMR (400.00 MHz,
CDCl3) d 8.12 (d, 2H, J 8.8 Hz), 8.10 (dd, 1H, J 0.8 Hz),
8.00 (dd, 1H, J 8.0, 1.6 Hz), 7.53 (td, 1H, J 7.6, 0.8 Hz),
7.50 (d, 2H, J 8.8 Hz), 7.25 (td, 1H, J 8.0, 1.6 Hz); 13C NMR
(100.0 MHz, CDCl3) d 175.6, 167.8, 141.8, 137.5, 133.1,
131.7, 129.2, 129.2, 128.9, 128.3, 125.3, 94.5; IR (KBr)

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Synthesis and Antibacterial Evaluation of 3,5-Diaryl-1,2,4-oxadiazole Derivatives
J. Braz. Chem. Soc.
n / cm-1 1581, 1535, 1404, 1357, 1087, 833, 740; HRMS
(ESI) m/z, calcd. for C14H8ClIN2O [M + H]+: 382.9443,
found: 382.9435.
m/z, calcd. for C14H7ClN4O5 [M + H]+: 347.0105, found:
347.0110.
3-(4-Chlorophenyl)-5-(2-chloro-5-nitrophenyl)-1,2,4-oxa-
3-(4-Chlorophenyl)-5-(3-bromophenyl)-1,2,4-oxadiazole
diazole (30)
1
(25)
Yield: 48%; mp 212-214 ºC; H NMR (400.00 MHz,
o
1
Yield: 47%; mp 159-161 C; H NMR (400.00 MHz,
CDCl3) d 8.35 (s, 1H), 8.13 (d, 1H, J 8.0 Hz), 8.11 (d, 2H,
J 8.0 Hz), 7.75 (d, 1H, J 8.0 Hz), 7.49 (d, 2H, J 8.0 Hz),
7.43 (t, 1H, J 8.0 Hz); 13C NMR (100.0 MHz, CDCl3)
d 174.5, 168.3, 137.5, 135.8, 131.0, 130.7, 129.2, 128.8,
126.6, 125.9, 125.2, 123.2; IR (KBr) n / cm-1 1597, 1550,
1473, 1404, 1357, 840, 748, 671; HRMS (ESI) m/z, calcd.
for C14H8BrClN2O [M + H]+: 334.9509, found: 334.9512.
CDCl3) d 9.06 (sd, 1H, J 2.4 Hz), 8.39 (dd, 1H, J 8.8,
2.4 Hz), 8.15 (d, 2H, J 8.4 Hz), 7.82 (d, 1H, J 8.8 Hz), 7.53
(d, 2H, J 8.4 Hz); 13C NMR (100.0 MHz, CDCl3) d 172.6,
168.2, 146.5, 140.5, 137.9, 132.8, 129.4, 129.0, 127.3,
127.1, 124.7, 124.7; IR (KBr) n / cm-1 1527, 1411, 1350,
1095, 833, 732; HRMS (ESI) m/z, calcd. for C14H7Cl2N3O3
[M + H]+: 335.9864, found: 335.9868.
3-Phenyl-5-(2-nitrophenyl)-1,2,4-oxadiazole (31)
o
1
3-(4-Chlorophenyl)-5-(2-nitrophenyl)-1,2,4-oxadiazole (26)
Yield: 47%; mp 87-89 C; H NMR (600.00 MHz,
CDCl3) d 8.15 (d, 2H, J 6.6 Hz), 8.04 (m, 2H), 7.80 (m,
2H), 7.53 (m, 3H); 13C NMR (150.0 MHz, CDCl3) d 172.6,
169.2, 149.0, 133.2, 133.1, 131.7, 131.5, 129.1, 127.8,
126.5, 124.8, 119.3; IR (KBr) n / cm-1 1527, 1441, 1355,
1300, 910, 851, 791, 723, 687, 579; HRMS (ESI) m/z,
calcd. for C14H9N3O3 [M + H]+: 268.0644, found: 268.0651.
o
1
Yield: 51%; mp 137-139 C; H NMR (400.00 MHz,
CDCl3) d 8.08 (d, 2H, J 8.0 Hz), 8.03 (m), 7.80 (m), 7.48
(d, 2H, J 8.0 Hz); 13C NMR (100.0 MHz, CDCl3) d 172.6,
168.2, 148.8, 137.7, 133.2, 132.9, 131.4, 129.3, 128.9,
124.8, 124.7, 118.9; IR (KBr) n / cm-1 1555, 1527, 1481,
1404, 1350, 1095, 840, 740; HRMS (ESI) m/z, calcd. for
C14H8ClN3O3 [M + H]+: 302.0326, found: 302.0327.
3-(3-Chlorophenyl)-5-(2-nitrophenyl)-1,2,4-oxadiazole (32)
o
1
3-(4-Chlorophenyl)-5-(3-nitrophenyl)-1,2,4-oxadiazole (27)
Yield: 50%; mp 95-97 C; H NMR (400.00 MHz,
CDCl3) d 8.13 (t, 1H, J 1.6 Hz), 8.04 (m, 3H), 7.81 (m,
2H), 7.52 (dt, 1H, J 8.0, 1.2 Hz), 7.45 (t, 1H, J 7.6 Hz);
13C NMR (100.0 MHz, CDCl3) d 172.8, 168.0, 148.7, 135.0,
133.2, 133.0, 131.6, 131.4, 130.3, 128.0, 127.7, 125.7,
124.7, 118.9; IR (KBr) n / cm-1 1529, 1434, 1355, 1340,
768, 744, 683; HRMS (ESI) m/z, calcd. for C14H8ClN3O3
[M + H]+: 302.0327, found: 302.0260.
o
1
Yield: 45%; mp 168-170 C; H NMR (400.00 MHz,
CDCl3) d 9.06 (s, 1H), 8.53 (d, 1H, J 7.6 Hz), 8.48 (d, 1H,
J 7.6 Hz), 8.13 (d, 2H, J 8.8 Hz), 7.79 (t, 1H, J 8.0 Hz), 7.51
(d, 2H, J 8.4 Hz); 13C NMR (100.0 MHz, CDCl3) d 173.7,
168.5, 148.7, 137.8, 133.5, 130.5, 129.3, 128.9, 127.2,
125.7, 124.8, 123.2; IR (KBr) n / cm-1 1600-1400, 1527,
1350, 1273, 740; HRMS (ESI) m/z, calcd. for C14H8ClN3O3
[M + H]+: 302.0327, found: 302.0324.
3-(3-Pyridyl)-5-phenyl-1,2,4-oxadiazole (33)
o
1
3-(4-Chlorophenyl)-5-(4-nitrophenyl)-1,2,4-oxadiazole (28)
Yield: 64%; mp 194-196 C; H NMR (400.00 MHz,
CDCl3) d 8.99 (d, 1H, J 2.0 Hz), 8.75 (dd, 1H, J 4.8, 1.6 Hz),
8.15 (dt, 1H, J 8.0, 2.0 Hz), 8.10 (d, 2H, J 7.2 Hz), 7.62 (t,
1H, J 7.6 Hz), 7.50 (t, 2H, J 8.0 Hz), 7.40 (m, 1H); 13C NMR
(100.0 MHz, CDCl3) d 163.8, 154.9, 152.2, 147.7, 134.8,
133.2, 129.5, 129.3, 128.6, 127.4, 123.6; IR (KBr) n / cm-1
3421, 3327, 3064, 1724, 1632, 1604, 1561, 1448, 1396,
1263, 1090, 1067, 1023, 930, 814, 698; HRMS (ESI) m/z,
calcd. for C13H9N3O [M + H]+: 224.0746, found: 224.0751.
o
1
Yield: 45%; mp 154-156 C; H NMR (400.00 MHz,
CDCl3) d 8.41 (m, 4H), 8.12 (d, 2H, J 8.4 Hz), 7.51 (d, 2H,
J 8.8 Hz); 13C NMR (100.0 MHz, CDCl3) d 173.8, 168.6,
150.3, 137.8, 129.3, 129.2, 129.2, 128.9, 124.8, 124.4;
IR (KBr) n / cm-1 1580, 1559, 1519, 1407, 1347, 1091,
840, 742, 715; HRMS (ESI) m/z, calcd. for C14H8ClN3O3
[M + H]+: 302.0327, found: 302.0328.
3-(4-Chlorophenyl)-5-(3,5-dinitrophenyl)-1,2,4-oxadiazole
(29)
3-(3-Pyridyl)-5-(2-chlorophenyl)-1,2,4-oxadiazole (34)
1
o
1
Yield: 47%; mp decomp.; H NMR (400.00 MHz,
CDCl3) d 9.17 (sd, 2H, J 2.4 Hz), 9.10 (st, 1H, J 2.0 Hz),
8.20 (d, 2H, J 8.8 Hz), 7.73 (d, 2H, J 8.8 Hz); C NMR
(100.0 MHz, CDCl3) d 173.1, 168.3, 149.1, 137.3, 130.0,
129.5, 128.7, 128.2, 126.3, 124.8; IR (KBr) n / cm-1 1598,
1537, 1469, 1405, 1338, 1095, 853, 763, 720; HRMS (ESI)
Yield: 52%; mp 83-85 C; H NMR (600.00 MHz,
CDCl3) d 9.42 (s, 1H), 8.78 (dd, 1H, J 4.8, 1.2 Hz), 8.48
(dt, 1H, J 8.4, 1.2 Hz), 8.18 (dd, 1H, J 7.8, 1.2 Hz), 7.62
(dd, 1H, J 8.4, 0.6 Hz), 7.55 (td, 1H, J 7.8, 1.2 Hz), 7.47
13
13
(m, 2H); C NMR (150.0 MHz, CDCl3) d 174.8, 166.7,
152.0, 148.6, 134.9, 134.0, 133.3, 131.9, 131.5, 127.1,

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Cunha et al.
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123.7, 123.3, 123.1; IR (KBr) n / cm-1 1595, 1583, 1407,
1364, 739, 700; HRMS (ESI) m/z, calcd. for C13H8ClN3O
[M + H]+: 258.0429, found: 258.0433.
2H, J 7.2 Hz), 7.53 (m, 3H), 7.16 (d, 1H, J 6.4 Hz), 7.06
(t, 1H, J 6.0 Hz); 13C NMR (100.0 MHz, CDCl3) d 174.7,
166.5, 158.3, 138.1, 135.6, 129.6, 129.0, 128.1, 124.5,
120.4, 118.1, 108.2; IR (KBr) n / cm-1 3166, 3133, 1625,
1593, 1551, 1482, 1406, 1362, 1231, 1088, 830, 751,
658; HRMS (ESI) m/z, calcd. for C14H9ClN2O2 [M + H]+:
273.0353, found: 273.0358.
3-(3-Pyridyl)-5-(2-bromophenyl)-1,2,4-oxadiazole (35)
o
1
Yield: 45%; mp 90-92 C; H NMR (600.00 MHz,
CDCl3) d 9.43 (s, 1H), 8.80 (d, 1H, J 4.2 Hz), 8.48 (dt, 1H,
J 7.8, 1.8 Hz), 8.12 (dd, 1H, J 7.8, 1.2 Hz), 7.83 (d, 1H,
13
J 7.8 Hz), 7.50 (m, 3H); C NMR (150.0 MHz, CDCl3)
Evaluation of biological activity
d 175.3, 166.7, 151.9, 148.6, 135.0, 134.9, 133.4, 132.2,
127.7, 125.3, 123.7, 123.2, 122.2; IR (KBr) n / cm-1 1599,
1407, 1366, 1024, 741, 702, 645; HRMS (ESI) m/z, calcd.
for C13H8BrN3O [M + H]+: 301.9923, found: 301.9924.
The antibacterial assay evaluated Gram-negative
(Escherichia coli American Type Culture Collection
(ATCC) 25922, Pseudomonas aeruginosa ATCC 27853 and
Proteus mirabilis ATCC 25933) and Gram-positive bacteria
(Staphylococcus aureus ATCC 25923 and Enterococcus
faecalis ATCC 29212). These microorganisms were first
placed in Mueller-Hinton broth and maintained for 24 h
at 37 oC.
The microbial suspensions were prepared by comparing
turbidity with the MacFarland scale standard No. 0.5 for
the preliminary determination of activity.21 The suspensions
in question were seeded by continuous induction in Petri
dishes containing Mueller-Hinton agar medium with the aid
of a swab. Then, 100 μM solution of each test compound,
in 20% DMSO, were added to the plate. There was one
plate for each bacterium being tested. The assays were
performed in duplicate.
3-(3-Pyridyl)-5-(2-iodophenyl)-1,2,4-oxadiazole (36)
o
1
Yield: 46%; mp 84-86 C; H NMR (400.00 MHz,
CDCl3) d 9.44 (d, 1H, J 2.0 Hz), 8.82 (dd, 1H, J 4.8, 1.6 Hz),
8.49 (dt, 1H, J 8.0, 1.6 Hz), 8.14 (d, 1H, J 8.0 Hz), 8.03
(dd, 1H, J 8.0, 1.6 Hz), 7.55 (t, 1H, J 7.6 Hz), 7.50 (m,
1H), 7.27 (td, 1H, J 7.6, 1.6 Hz); 13C NMR (100.0 MHz,
CDCl3) d 176.0, 166.6, 151.8, 148.5, 141.8, 135.1, 133.2,
131.8, 129.0, 128.5, 123.9, 123.3, 94.5; IR (KBr) n / cm-1
1705, 1581, 1405, 1366, 1017, 736, 697; HRMS (ESI) m/z,
calcd. for C13H8IN3O [M + H]+: 349.9785, found: 349.9786.
3-(3-Pyridyl)-5-(3,5-dinitrophenyl)-1,2,4-oxadiazole (37)
o
1
Yield: 45%; mp 137-139 C; H NMR (400.00 MHz,
DMSO-d6) d 9.32 (sd, 1H, J 1.6 Hz), 9.18 (sd, 2H,
J 2.0 Hz), 9.10 (st, 1H, J 2.0 Hz), 8.85 (dd, 1H, J 4.8,
1.6 Hz), 8.53 (dt, 1H, J 8.0, 2.0 Hz), 7.68 (t, 1H, J 4.8 Hz);
13C NMR (100.0 MHz, DMSO-d6) d 173.2, 167.4, 153.3,
149.2, 148.4, 135.4, 128.4, 126.3, 124.9, 122.9, 122.4; IR
(KBr) n / cm-1 1679, 1636, 1602, 1533, 1344, 918, 726,
704, 654; HRMS (ESI) m/z, calcd. for C13H7N5O5 [M + H]+:
314.0447, found: 314.0440.
The blank (negative) assay with 20% DMSO and the
positive control for antibacterial activity used the antibiotic
o
amoxicillin (100 μM). Plates were incubated at 37 C
for 24 h. After this period, the results were obtained by
observing the formation of the inhibition halos.21
MIC determination assays
In the assays to determine MIC, the compounds under
evaluation were used in different concentrations, obtained
by successive dilution of a stock solution.22 Concentrations
of 100, 75, 50 and 25 μM were initially used to define the
range at which the minimum inhibitory concentration was
localized. Subsequently, serial dilutions were made using
narrower concentration ranges.
Control assays were: (i) with Mueller-Hinton broth, 20%
DMSO solution and bacterial suspension; (ii) only broth
and 20% DMSO solution; (iii) with 20% DMSO solution,
broth and the test substance; and (iv) with a commercial
antibiotic solution of amoxicillin and broth with bacteria.
All assays were performed at the same concentrations
described above for the synthesized products. Observation
of turbidity in the wells of the plate was done with the naked
eye. All assays were performed in triplicate.
3-(3-Pyridyl)-5-(3-nitrophenyl)-1,2,4-oxadiazole (38)
o
1
Yield: 46%; mp 142-144 C; H NMR (400.00 MHz,
CDCl3) d 9.06 (s, 1H), 8.54 (d, 1H, J 7.6 Hz), 8.49 (d, 1H,
J 8.0 Hz), 8.17 (s, 1H), 8.07 (d, 1H, J 7.2 Hz), 7.80 (t, 1H,
J 8.0 Hz), 7.53 (d, 1H, J 7.6 Hz), 7.46 (t, 1H, J 7.6 Hz);
13C NMR (100.0 MHz, CDCl3) d 173.8, 168.3, 148.7,
135.1, 133.5, 131.6, 130.5, 130.3, 128.0, 127.7, 127.2,
126.7, 125.6, 123.2; IR (KBr) n / cm-1 1623, 1525, 1409,
1345, 731, 678; HRMS (ESI) m/z, calcd. for C13H8N4O3
[M + H]+: 269.0596, found: 269.0670.
3-(4-Chlorophenyl)-5-(2-hydroxyphenyl)-1,2,4-oxadiazole
(39)
o
1
Yield: 45%; mp 156-158 C; H NMR (400.00 MHz,
CDCl3) d 10.40 (s, 1H), 8.07 (d, 2H, J 6.8 Hz), 8.01 (d,

2410
Synthesis and Antibacterial Evaluation of 3,5-Diaryl-1,2,4-oxadiazole Derivatives
J. Braz. Chem. Soc.
Results and Discussion
and 33) derived from p-chlorobenzonitrile and 3-cyano-
pyridine, respectively, were synthesized as template
molecules of each set, as shown in Scheme 2.
Synthesis of the 1,2,4-oxadiazol derivatives (20-39)
From compound 21, a series with different halogen
atoms at the ortho position of the aromatic ring attached
to the C-5 heterocycle was synthesized (Figure 4). This
selection is associated with the importance of halogens
as pharmacophoric groups present in the structure of
antimicrobial compounds.40 The purpose of this series was
to evaluate the influence of halogen atoms on the C-ring.
Compounds 22, 23, 24 substituted with –Cl, –Br and –I
in the ortho position of the C-ring were synthesized. In
order to investigate the influence of the substituent position
on the bioactivity, the meta-brominated (25) isomer was
synthesized.
The literature classifies the nitro (–NO2) substituent as
a pharmacophore of great importance in the chemistry of
antibiotics, which is also present in the structure of known
antibacterials, such as chloramphenicol.41,42 Thus, the three
nitrated isomers (26, 27, 28) were synthesized (Figure 5) in
order to investigate the influence of this substituent.
In addition, two new derivatives containing two nitro
groups (29) and one chlorinated-nitrated compound (30)
were synthesized (Figure 5). The insertion of the second
nitro group in 10 had as main goal to increase the inhibition
efficiency, while that of the chlorine atom aimed to favor
a higher lipophilicity, considering that this parameter
influences the antibacterial activity.40 Following the same
strategy, a compound containing the C-ring mononitrated
and no substituents onA-ring (31) was synthesized in order
to check whether the absence of the chlorine atom would
influence the potential bioactivity. Finally, a compound with
a meta-chlorinated substitution pattern on the ring A (32)
was synthesized to evaluate the effect of the change of
position of the chlorine.
Initially, the treatment of the nitrile derivatives with
hydroxylamine hydrochloride produced the respective
amidoximes in yields of about 98%, which reacted with
the carboxylic acid chlorides yielding the 1,2,4-oxadiazole
nuclei (Scheme 1).
According to the literature, derivatives containing
homoaromatic rings23-26 and heteroaromatic13 compounds,
such as benzene and pyridine, exhibit antimicrobial
properties. Thus, new compounds have been synthesized
using the concept of molecular hybridization27 by combining
pharmacophore groups of different structures. Some
substitution patterns were adopted for both aromatic rings
based on structures reported in the literature (Figure 3).
Based on this information, twenty oxadiazole
derivatives were synthesized using benzonitrile (1),
4-chlorobenzonitrile (2), 3-chlorobenzonitrile (3), and
3-cyanopyridine (4). This strategy aimed to investigate the
influence of the different substituents and their positions on
the bioactivity of the synthesized products. The synthesis
strategy employed carboxylic acids containing halogens
and nitro groups as substituents at different positions
(Table 1).
The compounds were synthesized based on the
structural skeleton reported in Table 1,25,26,39 which has the
central 1,2,4-oxadiazole nucleus and phenyl radicals linked
at the C-3 and C-5 positions of the heterocycle. Thus, two
sets of compounds involving chlorinated and pyridine
derivatives were synthesized.
Initially, for the comparative effect of the two sets
of molecules, a basic compound without any type of
substitution was synthesized (20). Two compounds (21
Figure 3. Examples of antimicrobial oxadiazole derivatives.

Vol. 29, No. 11, 2018
Cunha et al.
2411
Table 1. Structure, yield and melting points of synthesized heterocycles
Substituent
Compound
Yield / %
Melting pointa / oC
R1
–
R2
H
R3
X
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
C
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
–
76
68
49
42
40
45
51
44
38
47
44
43
40
64
52
38
41
43
46
45
108-110 (110)28
111-113 (112)29
128-130 (128)30
130-134
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
–
H
–
2-Cl
2-Br
2-I
–
–
–
91-93
3-Br
2-NO2
3-NO2
4-NO2
3-NO2
2-Cl
2-NO2
2-NO2
H
–
159-161
–
137-139
–
168-170 (168)31
154-156 (154-156)32
decomposed
212-214 (213)33
87-89 (88-90)34
95-97
–
5-NO2
5-NO2
–
3-Cl
–
–
–
194-196 (196)35
83-85 (86)36
90-92
–
2-Cl
2-Br
2-I
–
–
–
–
–
84-86
–
3-NO2
3-NO2
2-OH
5-NO2
137-139
–
–
–
142-144 (142-144)37
156-158 (156-157)38
4-Cl
aCompounds without the reference value in parentheses are unpublished.
Scheme 2. General design strategy for the compound sets and series.
Figure 4. Oxadiazole derivatives containing aryl halides bound to the C-5 of the heterocycle ring.

2412
Synthesis and Antibacterial Evaluation of 3,5-Diaryl-1,2,4-oxadiazole Derivatives
J. Braz. Chem. Soc.
Figure 5. Oxadiazole derivatives containing phenyl nitro radical bonded to the C-5 of the heterocycle.
Incontinuity, sixcompoundscontainingaheteroaromatic
substituent were synthesized using the strategy adopted
for the first set of compounds. The synthesis strategy
employed cyano-pyridine, since the literature reports that
1,2,4-oxadiazole rings containing the pyridyl radical as
substituent have antibacterial activity (Figure 6).13 The
substitution of the phenyl by a pyridyl radical, bound
to the C-3 of the heterocycle, has focused on increasing
the polarization of the compound, as well as enabling
hydrogen bonding interactions,40 which are relevant
within the biological context. In this manner, a standard
compound (33), which did not have substituents on
the aromatic rings, was synthesized. In the following,
different halogens (–Cl, –Br and –I) were inserted onto the
homoaromatic ring. The products 34-36 were synthesized
in order to compare these compounds with their analogs
synthesized in the first group (Figure 6).
Assays for antibacterial activity
Table 2 presents the preliminary results of the
antibacterial properties of the synthesized compounds
using the agar dilution technique. These assays were
conducted using a concentration of 100 μM. This value
was established as the desired activity parameter as other
studies using concentrations close to this value were found
in the literature.43
The results in Table 2 indicate that some of the
compounds synthesized were able to inhibit the bacteria
P. mirabilis, E. faecalis and E. coli. However, none of
the compounds showed activity against S. aureus and
P. aeruginosa.
Compounds 23, 26, 27, 28, 30, 32 and 38, which
showed inhibition of the three bacteria mentioned above,
were selected for determination of the MIC values. Serial
dilutions of the solutions of the compounds together with
the broth containing the bacteria analyzed in this step were
conducted for these assays. First, a wide concentration
range was used (100, 75, 50 and 25 μM), and then gradually
narrowed. Table 3 shows the results of the MIC values
found for the assays with the bacteria P. mirabilis, E. coli
and E. faecalis.
Figure 6. Oxadiazole derivatives containing pyridyl radical bound to the
C-3 of the heterocycle.
The results of the MIC assays (Table 3) show that
compounds 26 and 30 provided the best results, followed
by compound 27. These compounds showed MIC values
around 60, 62 and 68 μM for E. coli, respectively, and they
presented values of 72, 72, and 76 μM for P. mirabilis,
In a similar way to the set of chlorinated compounds,
two nitrated (37 and 38) compounds and one containing a
hydroxyl (activator) group (39) were synthesized to verify
the influence of these groups on the bioactivity (Figure 7).
Figure 7. Oxadiazole derivatives containing nitro and hydroxyl groups as substituent.

Vol. 29, No. 11, 2018
Cunha et al.
2413
Table 2. Results of preliminary activity assays
growth. Also, the position of this substituent results in a
small influence on the final activity of the compound.
The results obtained for compounds 27 and 38 with
p-chlorophenyl and pyridyl radicals, respectively, attached
to the C-3 of the heterocycle showed that compound 27 had
better inhibition efficiency.
Inhibition capacity of compound 26, which contains
a chlorine atom at the para position, provided the best
result compared to 32, containing the same halogen atom
at the meta position. A similar result was observed for
compounds 23 and 25, where the latter was inactive,
confirming that the change in position of the substituent
decisively affected the activity.
The biological results of the heterocycles were
correlated with the physicochemical data (solubility
parameter (log P) and the polar surface area (PSA)), which
were obtained using the online software Molinspiration.45
The results for all compounds are shown in Table 4.
Correlating the MIC values and physicochemical data
for compounds 26, 27 and 28, where the nitro groups are
in different positions, inhibition efficiencies in the order
ortho (26) > meta (27) > para (28) were observed. The
physicochemical parameters for these three compounds
showed very close values for log P and PSA. Therefore,
it is assumed that these factors should not be considered
responsible for the different bioactivities found, and the
ratio of observed differences is probably a spatial question.
Compounds 27 and 30 have the same structure, except
for the insertion of the chlorine atom at the C-2-position
of the C-ring in compound 30, which showed slightly
better bacterial inhibition efficiency. This may have been
influenced by the higher lipophilicity of this compound,
as observed by the log P values in Table 4. The results of
Table 3 indicate that compound 31 showed low antibacterial
activity against all bacteria. This factor suggests that the
presence of the chlorine atom in the A ring influences the
activity.
Bacteriuma
Compound
P. mirabilis
E. faecalis
E. coli
20
–
–
–
+
–
–
+
+
+
–
+
+
+
–
–
–
–
–
+
–
+
–
–
–
+
–
–
+
+
+
–
+
–
–
–
–
–
–
–
–
–
+
–
–
–
+
–
–
+
+
+
–
+
+
+
–
–
–
–
–
+
–
+
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Amoxicillin
aP. aeruginosa and S. aureus were resistant to the synthesized compounds.
The signals indicate that the bacteria were sensitive (+) or resistant (–).
respectively. All three compounds had lower inhibition
efficiency (ca. 100 μM) against E. faecalis.
Comparing the structures of the eight heterocycles that
showed activity against the evaluated microorganisms,
seven of them contained the nitro group, only compound 23
did not. The results in Table 3 indicate that the nitro group
provided an improved inhibition efficiency compared to the
compound (23) independently of its position on the ring.
The analysis of the results for compounds 26, 27 and 28,
where the nitro substituents are in different positions (o, m,
p) suggests that the nitro group favors inhibition of bacterial
The only active compound (38), in the pyridine set,
showed low antibacterial activity. It is a mononitrated
compound, which, according to Table 4, has a log P = 2.59,
considerably lower than the compounds with the higher
Table 3. Minimum inhibitory concentration (MIC) values for the bioactive synthesized heterocycles
MICa / μM
Bacterium
Compound
23
86
26
72
95
60
27
76
28
86
30
72
31
100
–
32
98
–
38
95
–
Amoxicillin
P. mirabilis
E. faecalis
E. coli
4
16
2
100
82
100
68
100
78
100
62
100
93
95
aMinimum inhibitory concentration (MIC) values represent the average of three readings with uncertainty about 5%.44

2414
Synthesis and Antibacterial Evaluation of 3,5-Diaryl-1,2,4-oxadiazole Derivatives
J. Braz. Chem. Soc.
Table 4. Determination of the values of the solubility parameter (log P)
mechanism, which results in the formation of free radicals
with preferential toxicity to bacterial and parasitic cells.
The nitro group is reduced by an electron transfer protein
in the bacterium. The reduced nitro compound may cause
deoxyribonucleic acid (DNA) spiral rupture. Mammalian
cells remain unharmed as they lack the enzymes to reduce
the nitro group.47
and polar surface area (PSA) of the synthesized compounds45
Compound
20
log P
3.73
4.40
5.03
5.17
5.44
5.19
4.32
4.34
4.36
4.25
4.97
3.64
4.29
2.65
3.29
3.42
3.69
2.50
2.59
4.14
PSA
38.92
38.92
38.92
38.92
38.92
38.92
84.75
84.75
84.75
130.57
84.75
84.75
84.75
51.82
51.82
51.82
51.82
143.46
97.64
59.15
21
22
23
The transmembrane passageway of these molecules
occurs by passive diffusion and increases as the free
radicals from the bioreduction process destabilize the cell
membrane. Thus, due to the increase in the intracellular
concentration of nitro compounds, a greater number of
free radicals can be generated and, consequently, greater
damage is caused by oxidative stress48 (Scheme 3).
Biological activation through the bioreduction process
of nitro compounds is a target to improve the biological
activity of this class of compound. Thus, Rozenski et al.49
were able to relate the antibacterial action with the potential
of reducing some nitrocompounds, proving that this
bioactivity is due to the reduction of the nitro group.49,50
The formation of free radicals from the bioreduction
process of the nitro group can result in peroxidation of
biological membranes and proteins, inhibition of enzymes
and damage to DNA. One of the processes involved
in the bioreduction of the nitro group in anaerobic and
sometimes aerobic microorganisms is the interruption of
the normal flow of electrons from the respiratory chain,
which interferes with carbohydrate degradation, proton
transposition and energy production.51
This mechanism could occur by inhibiting some
enzymes belonging to the citric acid cycle involved in the
formation of acetyl coenzyme A (CoA), CoA-SH from
pyruvic acid, and in the subsequent process of cellular
respiration. The enzymes of the pyruvate complex of
microorganisms are responsible for the reduction of
some nitrocompounds promoting the formation of free
radicals (Ar–NO2•– and OH•) and the interruption of the
physiological flow of electrons. The lethal event, however,
is not the inhibition of respiration, but the formation of toxic
radicals from oxidative stress.41
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
log P: solubility parameter; PSA: polar surface area.
activities. The low log P value indicates that this compound
is less lipophilic and possibly this physicochemical feature
affects its bioactivity, since low lipophilicity may be
associated with a greater difficulty of diffusion through
bacterial membranes.46
Compounds 29 and 37 have two nitro groups in
their structure, however, they were inactive. This fact,
at first glance, could indicate a contradiction, since the
mononitrated compounds were active. However, on
analyzing Table 4 it is apparent that the PSA values for 29
and 37 are considerably higher, suggesting a higher polarity
in relation to the mononitrated ones. This result may again
indicate a greater diffusion difficulty of these compounds
through bacterial membranes, which could explain their
lack of activity.46
Conclusions
The reason for the higher activity of the nitro compounds
could be associated with the easy reduction of the nitro
group, which is linked to its strong acceptance of electrons.
Nitrated compounds can act via the enzymatic bioreduction
This work has demonstrated that a classical and easy-to-
execute synthetic methodology can generate 1,2,4-oxadiazole
derivatives active against certain strains of pathogenic
bacteria. Some advantages of this synthetic method are the
Scheme 3. Bioreduction of nitro compounds.

Vol. 29, No. 11, 2018
Cunha et al.
2415
availability of the starting materials and easy isolation of
the compounds using silica column chromatography. The
biological evaluation showed that the nature of the substituent
groups, as well as their position on the aromatic ring of the
compounds affected minimum inhibition concentration
values. The results showed a possible pathway for the
synthesis of new 1,2,4-oxadiazole bioactive derivatives.
12. Gobec, M.; Tomasic, T.; Markovic, T.; Mlinaric-Rascan, I.;
Dolenc, M. S.; Jakopin, Z.; Chem.-Biol. Interact. 2015, 240,
200.
13. Karad, S. C.; Purohit, V. B.; Thummar, R. P.; Vaghasiya, B. K.;
Kamani, R. D.; Thakor, P.; Thakkar, V. R.; Thakkar, S. S.; Ray,
A.; Raval, D. K.; Eur. J. Med. Chem. 2017, 126, 894.
14. Parra, M.; Hidalgo, P.; Carrasco, E.; Barberá, J.; Silvino, L.;
Liq. Cryst. 2006, 33, 875.
Supplementary Information
15. Tomi, I. H. R.; J. Saudi Chem. Soc. 2012, 16, 153.
16. Guo, J.; Hua, R.; Sui, Y.; Cao, J.; Tetrahedron Lett. 2014, 55,
1557.
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
17. Pace, A.; Pierro, P.; Org. Biomol. Chem. 2009, 7, 4337.
18. Kivrac, A.; Zora, M.; Tetrahedron 2014, 70, 817.
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Acknowledgments
We thank the agencies CAPES and CNPq for the
financial support. The authors would like to thank Prof Dr
Rafael Garrett (Laboratório deApoio ao Desenvolvimento
Tecnológico (LADETEC)) for the HRMS and Fundação
Oswaldo Cruz for the NMR and IR spectra.
21. Ostrosky, E. A.; Mizumoto, M. K.; Lima, M. E. L.; Kaneko,
T. M.; Nishikawa, S. O.; Freitas, B. R.; Rev. Bras. Farmacogn.
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Submitted: April 5, 2018
Published online: June 20, 2018
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