Preparation and antibacterial properties of substituted 1,2,4-triazoles

Article abstract of DOI:10.1155/2015/879343

Background. Both 1,2,3- and 1,2,4-triazoles are nowadays incorporated in numerous antibacterial pharmaceutical formulations. Aim. Our study aimed to prepare three substituted 1,2,4-triazoles and to evaluate their antibacterial properties. Materials and Methods. One disubstituted and two trisubstituted 1,2,4-triazoles were prepared and characterised by physical and spectroscopic properties (melting point, FTIR, NMR, and GC-MS). The antibacterial properties were studied against three bacterial strains: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853), by the agar disk diffusion method and the dilution method with MIC (minimal inhibitory concentration) determination. Results. The spectroscopic characterization of compounds and the working protocol for the synthesis of the triazolic derivatives are described. The compounds were obtained with 15-43% yields and with high purities, confirmed by the NMR analysis. The evaluation of biological activities showed that the compounds act as antibacterial agents against Staphylococcus aureus (ATCC 25923), while being inactive against Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Conclusions. Our results indicate that compounds containing 1,2,4-triazolic moiety have great potential in developing a wide variety of new antibacterial formulations.

Full text of DOI:10.1155/2015/879343

Hindawi Publishing Corporation
Journal of Chemistry
Volume 2015, Article ID 879343, 5 pages
http://dx.doi.org/10.1155/2015/879343
Research Article
Preparation and Antibacterial Properties of
Substituted 1,2,4-Triazoles
Ionuu Ledeui,1 Vasile Bercean,2 Anda Alexa,3 Codruua Foica,1
Lenuua-Maria Futa,1 Cristina Dehelean,1 Cristina Trandafirescu,1
Delia Muntean,3 Monica Licker,3 and Adriana FuliaG1
1Faculty of Pharmacy, University of Medicine and Pharmacy “Victor Babe¸s”, 2 Efimie Murgu Square, 300041 Timi¸soara, Romania
2Faculty of Industrial Chemistry and Environmental Engineering, Politehnica University Timi¸soara, 6 Carol Telbisz,
300001 Timi¸soara, Romania
3Faculty of Medicine, University of Medicine and Pharmacy “Victor Babe¸s”, 2 Efimie Murgu Square, 300041 Timi¸soara, Romania
Correspondence should be addressed to Vasile Bercean; vbercean@gmail.com
Received 14 January 2015; Revised 22 February 2015; Accepted 22 February 2015
Academic Editor: Teodorico C. Ramalho
Copyright © 2015 Ionu¸t Lede¸ti et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Both 1,2,3- and 1,2,4-triazoles are nowadays incorporated in numerous antibacterial pharmaceutical formulations.
Aim. Our study aimed to prepare three substituted 1,2,4-triazoles and to evaluate their antibacterial properties. Materials and
Methods. One disubstituted and two trisubstituted 1,2,4-triazoles were prepared and characterised by physical and spectroscopic
properties (melting point, FTIR, NMR, and GC-MS). e antibacterial properties were studied against three bacterial strains:
Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853), by the agar disk
diffusion method and the dilution method with MIC (minimal inhibitory concentration) determination. Results. e spectroscopic
characterization of compounds and the working protocol for the synthesis of the triazolic derivatives are described. e compounds
were obtained with 15–43% yields and with high purities, confirmed by the NMR analysis. e evaluation of biological activities
showed that the compounds act as antibacterial agents against Staphylococcus aureus (ATCC 25923), while being inactive against
Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Conclusions. Our results indicate that compounds
containing 1,2,4-triazolic moiety have great potential in developing a wide variety of new antibacterial formulations.
1. Introduction
According to this, we set our goal in the synthesis, char-
acterization (physical and spectroscopic properties, melting
point, FTIR, NMR, and GC-MS), and evaluation of biological
activity of three substitutes triazoles (1–3) against Staphylo-
coccus aureus (ATCC 25923), Escherichia coli (ATCC 25922),
and Pseudomonas aeruginosa (ATCC 27853).
Triazolic nucleus is nowadays considered an important moi-
ety in the design and synthesis of bioactive compounds that
are associated with numerous biological activities [1] such as
antibacterial, antifungal [2], anti-inflammatory [3], anticon-
vulsant [4], anti-HIV [5], antineoplastic, and antiproliferative
[6–13]. Additionally, there are review studies that indicate the
fact that 1,2,4-triazoles occupy a distinctive place in the field
of medicinal and pharmaceutical chemistry [14, 15], as well
as in industry [16]. Also, synthesis and complete character-
ization by both spectroscopic and thermal techniques were
reported in literature for numerous derivatives bearing 1,2,4-
triazole moieties [17–20].
2. Materials and Methods
2.1. Chemistry. e reagents were commercial products of
analytical purity (Chimopar, Merck, Fluka) and used as
received. Melting points were determined on a Bo¨etius
PHMK (Veb Analytik, Dresden, Germany) instrument (the
values are uncorrected), and thin-layer chromatography

2
Journal of Chemistry
N
N
SH
H3C
i
ii
N
C6H5-NH2
C2H5-NH-CS-NH-NH2
C6H5
(4)
(1)
i = (1) CS2/KOH/C2H5OH-HO ∼20∘C; (2) ClCH2COONa/H2O ∼20∘C; (3) H2N-NH2·H2O reflux
ii = (1) CH3COOC2H5 (b)/CH3ONa/CH3OH reflux; (2) HCl dil
Scheme 1: e synthesis of 4H-3-mercapto-5-methyl-4-phenyl-1,2,4-triazole (1).
H
N
N
i
ii
H2N-CS-NH-NH2
H2N-CS-NH-NH-CO-C6H5
C6H5
SH
N
(5)
(2)
i = C6H5-COCl/Py/DMF ∼20∘C; ii = (1) NaOH/H2O-C2H5OH reflux; (2) HCl dil.
Scheme 2: e synthesis of 1H-5-mercapto-3-phenyl-1,2,4-triazole (2).
(TLC) was carried out on silica gel-coated plates 60 F254
Merck using hexane : ethyl acetate 1 : 1 (v/v) as eluant.
FTIR spectra were recorded in KBr pellet using a
Specac Pellet Press (Specac Ltd., Kent, UK) on a Jasco
FT/IR-410 spectrophotometer (Jasco Analytical Instruments,
Easton, USA) using the following abbreviation for the
aspect/intensities of the bands: br: broad; s: strong; m:
medium; w: weak.
3097m; 3054m; 2908m; 1579m; 1498s; 1326s; 1288m; 1018w;
771w; 692m; 1H-NMR ꢁ (DMSO-ꢀ6, 200 MHz):13.50 (br.s,
H
1H, -SH); 7.30–7.65 (m, 5H, -C H ); 2.08 (s, 3H, -CH ).
6
5
3
1H-5-Mercapto-3-phenyl-1,2,4-triazole (2) was obtained
by benzoylation of thiosemicarbazide with benzoyl chlorides,
followed by cyclisation of crude 1-benzoyl-thiosemicarbazide
(5) with NaOH in aqueous-alcoholic medium, using a
method previously used in our research group (Scheme 2)
[23].
1H-NMR and 13C-NMR spectra were recorded on a
Bruker Avance AC200 spectrometer (Bruker Biospin Inter-
national, Ag, Aegeristrasse, Switzerland) in DMSO-ꢀ6, using
TMS as reference; chemical shifs are reported in ppm and the
coupling constants in Hz. e multiplicity of the signal and
the aspect of the band are abbreviated as follows: br: broad; s:
singlet; d: doublet; m: multiplet.
Compound 2, 1H-5-mercapto-3-phenyl-1,2,4-triazole,
was obtained as a white powder (yield 43%), TLC one spot,
m.p. 242–247∘C (lit. 254–256∘C [24]). FTIR (KBr, cm−1):
3450m; 3421m; 1630m; 1610m; 1563m; 1289m; 1080w; 776w;
1H-NMR ꢁ (DMSO-ꢀ6, 200 MHz): 13.87 (s, 1H, SH); 13.70
H
(s, 1H, NH); 7.47–7.95 (m, 5H, -Ph); 13C-RMN ꢁ (DMSO-ꢀ6,
100 MHz): 172.20 (C-S); 155.41 (C=N); 135.84 C(1ꢀ-C); 134.33
(4ꢀ-C); 130.86 (3ꢀ-C, 5ꢀ-C); 130.65 (2ꢀ-C, 6ꢀ-C).
Mass spectra GC-MS were obtained using an Agilent
G1701DA apparatus (Agilent Technologies, Inc., Santa Clara,
USA) using methanol as carrier solvent.
4H-4-Amino-5-(4-pyridyl)-3-mercapto-1,2,4-triazole (3)
was obtained using as starting compound, the hydrazide of
isonicotinic acid (6), using a previously described method by
us (Scheme 3) [25].
2.2. In Vitro Antibacterial Activity. e compounds were
screened for their antimicrobial activity against three bacte-
rial strains: Staphylococcus aureus (ATCC 25923), Escherichia
coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC
27853), by the agar disk diffusion method and the dilution
method with MIC (minimal inhibitory concentration) deter-
mination.
Compound 3, 4H-4-amino-5-(4-pyridyl)-3-mercapto-
1,2,4-triazole, was obtained as a white powder (yield 15%),
TLC one spot, m.p. 204–206∘C. GC-MS (M/z): purity 99%;
193(M; 100%), 105(Py-C=NH+, 27%); FTIR (KBr, cm−1):
3272s; 3162s; 1608m; 1318m; 826m; 736w; 1H-RMN ꢁ
H
(DMSO-ꢀ6, 200 MHz): 14.18 (s, 1H, -NH); 8.77 (d, 2H,
2.3. Experimental Protocols for the Synthesis of Bioac-
tive Compounds (1–3). e synthesis of 4H-3-mercapto-5-
methyl-4-phenyl-1,2,4-triazole (1) was carried out accord-
ing to a described protocol [21, 22] by heating 4-phenyl-
thiosemicarbazide (4) with ethyl acetate and sodium methox-
ide in methanol under reflux, followed by neutralization.
e synthesis of 4-phenylthiosemicarbazide was however
realized in a single-step reaction, by treating aniline with
J
ꢀ
ꢀ
= 5.6 Hz, 3ꢀH, 5ꢀH); 8.04 (d, 2H, J
ꢀ
ꢀ
= 5.6 Hz,
3 -H,5 H
2 -H,6 H
2ꢀH, 6ꢀH), 5.87 (s.broad, 2H, -NH ); 13C-RMN ꢁ (DMSO-
2
C
ꢀ6, 100 MHz): 167.6 (5-C); 150.07 (3ꢀC, 5ꢀC); 147.28 (3-C); 132.8
(1ꢀC); 121.48 (2ꢀC, 6ꢀC).
2.4. Disk Diffusion Method. e antimicrobial activity of the
compounds was evaluated according to the guidelines of
the National Committee for Clinical Laboratory Standards
(NCCLS, 1997) using agar disk diffusion method [26].
NCCLS recommends a bacterial suspension with a den-
sity equal to a 0.5 McFarland (which gives a final bacterial
concentration of 1-2⋅108 CFU/mL). e Mueller-Hinton agar
CS and alcoholic KOH solution, sodium chloroacetate, and
2
hydrazine hydrate under reflux (Scheme 1) [11].
Compound 1, 4H-3-mercapto-5-methyl-4-phenyl-1,2,4-
triazole, was obtained as a white powder (yield 21%), TLC
one spot, m.p. 214–215∘C. FTIR (KBr, cm−1): 3301m; 3158m;

Journal of Chemistry
3
N
N
i
CO-NH-NH2
N
SH
(3)
N
N
(6)
NH2
i = (1) CS2/KOH/H2O-C2H5OH; (2) H2N-NH2·H2O 2h r.t; (3) reflux 4h; (4) HCl dil
Scheme 3: e synthesis of 4H-4-amino-5-(4-pyridyl)-3-mercapto-1,2,4-triazole (3).
Table 1: Antimicrobial activity of compounds.
Zone of inhibition (mm)/MIC (ꢂg/mL)
Compounds
1
S. aureus ATCC 25923
0.5%–14 mm
1%–17 mm
1%–12 mm
1%–11 mm
E. coli
P. aeruginosa
40 ꢂg/mL
—
—
2
25 ꢂg/mL
—
—
—
—
3
25 ꢂg/mL
Gentamicin (10 ꢂg/disk)
20 mm
20 mm
19 mm
plates were inoculated with bacterial suspension using a
sterile cotton swab.
A series of solutions of the tested compounds were
prepared in DMSO, with a concentration of 0.01%, 0.1%,
0.5%, and 1%, respectively. Within 15 min afer the plates
were inoculated, sterile Whatman number 1 filter paper
disks (6 mm in diameter) impregnated with the solutions
tube with 50 ꢂL of bacterial suspension and 100 ꢂL of nutrient
medium.
We performed triplicate tests and, from all the tubes used
for MICs reading, cultures on Columbia agar supplemented
with 5% sheep blood were performed in order to verify the
results; no bacterial growth was recorded in the presence of
the tested solution to which MIC was attributed.
in DMSO of the tested compounds (20 ꢂL solution/disk,
corresponding to 2, 20, 100, and 200 ꢂg compound/disk, resp.,
for the four solutions prepared with each compound) were
distributed evenly on the surface, with at least 25 mm (center
to center) between them. Disks with gentamicin (10 ꢂg),
supplied by Bio-Rad, were used as positive control for the
antimicrobial activity. For negative control we used a paper
disk impregnated with dimethylsulfoxide (DMSO).
3. Results and Discussion
e synthesis of heterocyclic derivatives (1-3) was performed
according to indications mentioned in literature. e com-
pounds were obtained with moderate yields (15–43%) but
with high purity, fact confirmed by TLC analysis and evalua-
tion of melting interval. e formation of triazolic derivatives
was proven by FTIR and NMR analysis, as well by mass
spectrometry for the compound (3).
Plates inoculated with the bacterial suspensions were
incubated at 37∘C for 24 h. e inhibition zone diameters
were measured in millimeters, using a ruler. For all the
bacterial strains the disk diffusion tests were performed in
triplicate and the average reading was taken into account.
e results of the antimicrobial evaluation are presented
in Table 1.
e synthesized and evaluated derivatives with triazolic
structure were tested for antibacterial activity against three
bacterial strains by means of the disk diffusion method. e
2.5. Dilution Method. e MIC values were determined only
for the active compounds, with a zone of inhibition >10 mm.
results presented in Table 1 reveal that only two compounds
e MIC values were evaluated in the range of 1.56–50ꢂg/mL.
may act as anti-infective agents, inhibiting the growth of S.
aureus ATCC 25923 at concentrations within the range of
e MIC (ꢂg/mL) was determined by the binary microdi-
lution method. We prepared stock solutions in DMSO of the
tested compounds, at a concentration of 100 ꢂg/mL. From
these solutions, serial dilutions of the compounds (50, 25,
25–40 ꢂg/mL. It is worth mentioning that both compounds
are active against Gram-positive bacteria (S. aureus) while
no activity was present against Gram-negative bacteria (E.
12.5, 6.25, 3.12, and 1.56 ꢂg/mL) were prepared and brought,
coli, P. aeruginosa); these results demonstrate a specific
under aseptic conditions, to a final volume of 200 ꢂL with
nutrient medium. In all tubes 50 ꢂL of bacterial suspensions
were added, with a density equal to a 0.5 McFarland. All
tubes were incubated at 37∘C for 24 h. e MIC was recorded
as the minimum concentration of the compound which
inhibited the visible growth of the tested microorganisms.
For a negative control 50 ꢂL of DMSO was introduced in a
antimicrobial activity against Gram-positive bacterial infec-
tions. Gentamicin is a powerful broad-spectrum antibiotic,
largely used in clinical practice, usually in association with
ꢃ-lactam antibiotics, for the treatment of a wide range of
bacterial infections; in this paper, it served as reference for the
antimicrobial potency of the tested compounds. Both active
substances, as showed in Table 1, produced an inhibition zone

4
Journal of Chemistry
with a diameter between 11 and 17 cm at an MIC of 25–
40 ꢂg/mL. e results are comparable to the ones revealed in
case of gentamicin (20 mm), demonstrating the possibility of
future therapeutical use of the two compounds as potential
antibacterial agents.
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Conflict of Interests
e authors declare that there is no conflict of interests
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is work was supported by a grant from the University of
Medicine and Pharmacy “Victor Babe¸s” Timi¸soara (Grant II-
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