Synthesis and antimicrobial activities of some new 1,2,4-triazole derivatives

Article abstract of DOI:10.3390/molecules15042427

Some novel 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one (3, 6, 8, 9) derivatives and or 3-(4-methylphenyl)[1,2,4]triazolo[3,4-b][1,3]benzoxazole (5) were synthesized from the reaction of various ester ethoxycarbonylhydrazones (1a-e) with several p

Full text of DOI:10.3390/molecules15042427

Molecules 2010, 15, 2427-2438; doi:10.3390/molecules15042427
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antimicrobial Activities of Some New 1,2,4-Triazole
Derivatives
Hakan Bektaş 1, Nesrin Karaali 1, Deniz Şahin 1, Ahmet Demirbaş 1, Şengül Alpay Karaoglu 2,
Neslihan Demirbaş 1,*
1
Department of Chemistry, Karadeniz Technical University, 61080 Trabzon, Turkey
2
Department of Biology, Rize University, 53100 Rize, Turkey
∗ Author to whom correspondence should be addressed; E-Mail: neslihan@ktu.edu.tr
Received: 12 February 2010; in revised form: 9 March 2010 / Accepted: 22 March 2010 /
Published: 8 April 2010
Abstract: Some novel 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one (3, 6, 8, 9)
derivatives and or 3-(4-methylphenyl)[1,2,4]triazolo[3,4-b][1,3]benzoxazole (5) were
synthesized from the reaction of various ester ethoxycarbonylhydrazones (1a-e) with
several primary amines. The synthesis of 4-amino-5-(4-chlorophenyl)-2-[(5-mercapto-
1,3,4-oxadiazol-2-yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (13) was performed
starting from 4-Amino-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (2) by four
steps; then 13 was converted to the corresponding Schiff base (14) by using 4-
methoxybenzaldehyde. Finally, two Mannich base derivatives of 14 were obtained by
using morpholine or methyl piperazine as amine component. All newly synthesized
compounds were screened for their antimicrobial activities and some of which were found
to possess good or moderate activities against the test microorganisms.
Keywords: 1,2,4-Triazole; triazolo[3,4-b][1,3]benzoxazole; primary amine; Schiff base;
Mannich base
1. Introduction
The synthesis of high nitrogen containing heterocyclic systems has been attracting increasing
interest over the past decade because of their utility in various applications, such as propellants,
explosives, pyrotechnics and especially chemotherapy [1]. In the medicinal chemistry, azoles are
widely used and studied class of antimicrobial agents due to their safety profile and high therapeutic

Molecules 2010, 15
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index. Among these, Conazoles are a major class of azole-based drugs such as Itraconazole,
Fluconazole, Voriconazole, Ravuconazole etc. [2–5]. Some of other major applications of conazoles
are on crop protection. As pharmaceuticals, they are used for the treatment of local and systemic
fungal infections, which are important problems in phytopathology and especially in medicine, and
they are frequently observed in immune-compromised patients suffering from AIDS or subjected to
invasive surgery, anti-cancer therapy or graft receivers.
Several lines of evidence suggest that the primary target of azoles is the heme protein, which
cocatalyzes cytochrome P-450-dependent 14α-demethylation of lanosterol. Inhibition of 14
-
demethylase leads to depletion of ergosterol and accumulation of sterol precursors, including 14 -
methylated sterols (lanosterol, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol), resulting
in the formation of a plasma membrane with altered structure and function. The more recent triazole
derivatives, such as fluconazole, itraconazole, and voriconazole owe their antifungal activity at least in
part to inhibition of cytochrome P-450-dependent 14α-sterol demethylase [6].
The diseases caused by fungal species cause not only improve the cost of therapy but also may lead
mortality. Due to the inadequacy of alone standard antibiotic therapy in certain circumstances, more
efforts have been focused on addressing the problem of multidrug-resistant bacteria and the decreasing
of costs and consequences the obtained results from this [7–9]. Tuberculosis (TB) that is another
mortal infection, causes to death with approximately three million patients in the world every year.
According to the World Health Organization (WHO), about 30 million people will be infected within
next 20 years [10]. Thus, the treatment of infections has become an important and challenging problem
because of the increasing number of multi-drug resistant microbial pathogens [11]. In spite of a large
number of antibiotics and chemotherapeutics available for medical usage, the increasing resistance
made it necessary to continue the search for new antimicrobial substances. Though various molecules
designed and synthesized for this aim, the efforts have demonstrated that 1,2,4-triazoles and their
derivatives could be considered as possible antimicrobial agents, some of them studied in our
laboratories [12–24].
Moreover, synthesis of 1,2,4-triazoles fused to another heterocyclic ring has attracted wide spread
attention due to their diverse applications as antibacterial-, antidepressant-, antiviral-, antitumorial- and
anti-inflammatory agents, pesticides, herbicides dyes, lubricant and analytical reagents [25,26].
Among these, the commonly known systems are generally triazoles fused to pyridines, pyridazines,
pyrimidines, pyrazines and triazines. Although there are not many triazoles fused to thiadiazines or
thiadiazoles, a number of them are incorporated into a wide variety of therapeutically important
compounds possessing a broad spectrum of biological activities [26–29].
The condensation reaction of relatively small and linear molecules with suitable reagents is a
general method leading to the formation of heterocyclic systems [30]. For this purpose, ester
ethoxycarbonylhydrazones (1) are suitable precursors; a number of azole derivatives have been
obtained in our laboratory starting from compound 1 [12–21]. Other useful intermediates are the
compounds incorporating a hydrazide function in their structures [20,23].
In the present study, as a continuation of our studies on obtaining bioactive molecules, we have
performed the synthesis of some new 1,2,4-triazole derivatives and investigation of antimicrobial
activities of newly synthesized compounds (Scheme 1, Scheme 2).

Molecules 2010, 15
Scheme 1. Synthetic pathway for the preparaion of compounds 2–9.
2429
N
NH
OH
NH2
N
N
N
NH
R
O
R
N
N
O
R
O
N
OH
H2NNH2
NH2
NNHCO2Et
OEt
2: R= -C6H4CI(p-)
5: R= -C6H4CH3(p-)
4: R= -C6H4CH3(p-)
NH
R
N
1a:
R= -CH3
SO2
CI
CI
N
NH
N
N
N
NH
2
1b: R= -C6H4CI(p-)
1c: R= -C6H5
SO2CI
R
O
N
R
O
N
O
N
NH2
N
R
O
N
1d: R= -C6H4CH3(p-)
1e: R= -CH2C6H4CI(p-)
N
N
O
N
N
7: R= -CH3
6: R= -CH3
3: R= -C6H4CH3(p-)
N
NH
2
NH
2
N
N
N
H
NH
2
N
NH
N
NH
N
NH
R
O
N
R
O
R
O
N
N
N
N
N
H
9a: R= -CH3
9b: R= -CH2C6H4CI(p-)
8: R= -C6H5
Scheme 2. The synthetic routes for compounds 10–15.
O
O
N
N
N
NH
N
NH
NHNH2
SH
OEt
H2NNH2
O
N
N
N
N
N
N
O
N
O
OEt-
PhCHO
Cl
N
CS2/KOH
Cl
O
N
N
C
O
NH2
BrCH2CO2Et
N
O
N
C
N
Cl
NH2
NH2
H
Cl
Cl
H
10
13
12
2
11
(p-)CH3OC6H4CHO
R
O
N
N
N
N
NHNH2
N
N
S
O
SH
N
N
O
N
N
O
N
N
C
Mannich reactions
O
N
O
N
N
C
N
C
Cl
H
Cl
Cl
H
H
OCH3
OCH3
16
-N
N CH3
15a: R=
14
-N
O
15b: R=
2. Results and Discussion
The treatment of ester ethoxycarbonylhydrazones (1a-e) with various primary amines resulted in
the formation of 4,5-disubstitue-2,4-dihydro-3H-1,2,4-triazol-3-ones (3, 6, 8), while the reaction of 1d
with 2-hydroxyaniline afforded 3-(4-methylphenyl)[1,2,4]triazolo[3,4-b][1,3]benzoxazole (5). On the
other hand, the reaction of 1a,e with 1,4-di(3-aminopropyl)piperazine produced symmetric bitriazolyl-
4-ylpropyl piperazine derivatives (9a,b) (Scheme 1). Compounds 3, 5, 6, 8 and 9 exhibited NMR and
1
IR spectra consistent with their assigned structures. In the H NMR spectrum of 5, no signal
representing an -NH or -OH group was recorded. In addition, compound 5 gave elemental analysis and
mass spectral data consistent with the assigned structure (5). On the basis of these reported
observation, compound 5 was designated as 3-(4-methylphenyl)[1,2,4]triazolo[3,4-b][1,3]benzoxazole.

Molecules 2010, 15
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4-Amino-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (2) obtained from the reaction of 1c
with hydrazine hydrate in water at reflux temperature, crystallized from ethanol and gave IR
spectroscopic data consistent with literature [31].
The synthesis of ethyl [3-(4-chlorophenyl)-5-oxo-4-{[phenylmethylene]amino}-4,5-dihydro-1H-
1,2,4-triazol-1-yl]acetate (11) was performed by the reaction of compound 10 [27] with ethyl
bromoacetate in the presence of sodium ethoxide (Scheme 2). The reaction of compound 11 with
hydrazine hydrate in water afforded compound 12 but not 16. It was reported that the treatment of
Schiff bases derived from type 2 compounds might cause to the hydrolysis of alkylidenamino group to
free amino group via the attack of hydrazine hydrate (or water) to imine bond at the same time with the
1
attack to exocyclic ester group [23]. The H NMR spectrum of compound 12 showed complete
absence of signals relevant to structure 16. In addition, compound 12 gave M+1 ion peak in the mass
spectrum and good elemental analysis results.
The treatment of 12 with carbon disulphide in basic media caused to the conversion of hydrazide
side chain into 5-mercapto-1,3,4-thiadiazole ring, thus, compound 13 was obtained. It is known that
5-mercapto-1,3,4-oxadiazoles are exist as their mercapto-thioxo tautomeric forms [13–15]. As a result
of this tautomerism, the IR spectrum of 13 displayed two stretching bands, one of which observed at
2750 cm-1 belongs to –SH group, the other recorded at 1164 cm-1 represents the existent of -C=S
group. Moreover, compound 13 gave satisfactory NMR, mass and elemental analysis data.
The synthesis of compounds 15a,b was carried out by the Mannich reaction of compound 14 with
methyl piperazine (for 15a) or morpholine (for 15b) in the presence of formaldehyde solution.
Additional signals belonging to methyl piperazine or morpholine moiety and methylene linkage were
1
13
recorded at the related chemical shif values in the H and C NMR spectra of 15a,b. It was reported
that, the compounds having Schiff base structure may exist as E/Z geometrical isomers about the
–N=CH- double bond [12,19,33, 34]. The literature survey revealed that, the compounds containing
imine bond are present in higher percentage in dimethyl-d6 sulfoxide solution in the form of
geometrical E isomer about –N=CH- double bond [33]. The Z isomer can be stabilized in less polar
solvents by an intramolecular hydrogen bond. In the 1H NMR spectra of compounds 14 and 15a,b, the
-N=CH- signals were observed as two sets due to the existence of E and Z isomers.
None of the synthesized compounds showed antimicrobial activity against Candida tropicalis (Ct)
and Candida albicans (Ca). Among the compounds 4,5-disubstitue-2,4-dihydro-3H-1,2,4-triazol-3-
ones (3, 6, 8) and 9a,b; the compounds 3 and 8 exhibited moderate activities towards Escherichia coli
(Ec) and Klebsiella pneumoniae (Kp) which are containing a morpholine (for 3) or indol-3-ylethyl
moiety (for 8) in the position 4 of 1,2,4-triazol-3-one ring. Similarly, compound 11 having an imine
bond and compound 12 possesing hydrazide function in their structures showed moderate activities
against Enterobacter aerogenes (En), Staphylococcus aureus (Sa), Enterococcus faecalis (Ef) and
Bacillus careus (Be).
Good antimicrobial activities were found for compounds 13 and 14 against the test microorganisms,
which are including a 5-mercapto-1,3,4-oxadiazole ring bearing to 1,2,4-triazole nucleus via a
methylene linkage. The conversion of the amino group in position 4 of 1,2,4-triazole ring into
4-methoxyphenylenamino group caused no change in the antimicrobial activity for compound 14.
Among the Mannich bases of compound 14, 15b displayed good antimicrobial activities against the
test microorganisms that contain an additional morpholine moiety beside 1,2,4-triazole and 1,3,4-

Molecules 2010, 15
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oxadiazole rings, while other Mannich base, 15a, that has a methyl piperazine nucleus instead of
morpholine, exhibited good or moderate activities towards the test microorganisms except Escherichia
coli (Ec) and Klebsiella pneumoniae (Kp).
3. Experimental
3.1. General
Melting points were determined on a Büchi B-540 melting point apparatus and are uncorrected. 1H
NMR and 13C NMR spectra were recorded on a Varian-Mercury 200 MHz spectrometer. The IR
spectra were measured as potassium bromide pellets using a Perkin-Elmer 1600 series FTIR
spectrometer. Mass spectra were obtained at a Quattro LC-MS (ESI, 70 eV) Instrument (except
compounds 5, 9a, 9b and 15a). Combustion analysis was performed on a Costech Elementel
Combution System CHNS-O elemental analyzer. All the chemicals were obtained from Fluka Chemie
AG Buchs (Switzerland). Compounds 1a-e, 2 and 10 were prepared by the way reported earlier
[30–32].
3.2. General Method for the Synthesis of Compounds 3, 5, 6, 8, 9a,b
The corresponding compound 1 (10 mmol) was heated in an oil bath with a suitable primer amine
(represented in the scheme 1) (10 mmol for 3,5,6, 8; 20 mmol for 9a,b) at reflux temperature (between
90–120 ºC) for 2 h. After cooling to room temperature, 3–4 mL of ethyl acetate-petroleum ether
mixture was added to the viscous residue. On cooling the mixture in cold, a solid appeared. This crude
product was recrystallized from an appropriate solvent to afford the desired product.
5-(4-Methylphenyl)-4-morpholin-4-yl-2,4-dihydro-3H-1,2,4-triazol-3-one (3). Recrystallized from
ethyl acetate; yield 39%, m.p. 226–227 ºC. IR spectrum (KBr), ν, cm-1: 3200 (NH), 1699 (C=O), 1508
(C=N). 1H NMR spectrum (DMSO-d6), δ, ppm: 2.35 (s, 3H, CH3), 2.94 (brs, 4H, 2CH2), 3.81 (brs, 4H,
13
2CH2), 7.29 (d, 2H, arH), 7.45 (d, 2H, arH), 11.94 (s, 1H, NH). C NMR spectrum (DMSO-d6), δ,
ppm: 20.85 (CH3), 51.06 (2CH2), 66.15 (2CH2), arC: [123.69 (C), 127.25 (2CH), 128.87 (2CH),
139.52 (C)], 144.39 (triazole C3), 153.37 (triazole C5). MS (ESI): m/z (%) 283 (M +Na, 58), 261 (M+,
10), 230 (42), 218 (11), 208 (74), 140 (100), 118 (14), 112 (21). Anal. for: C13H16N4O2 Calcd. (%): C,
59.99, H, 6.20, N, 21.52. Found, (%): C, 60.18, H, 6.34, N, 21.23.
3-(4-Methylphenyl)[1,2,4]triazolo[3,4-b][1,3]benzoxazole (5). Recrystallized from ethanol; yield 70%,
1
m.p. 116–117 ºC. IR spectrum (KBr), ν, cm-1: 1621 (C=N), 1555 (C=N), 1243 (C-O). H NMR
spectrum (DMSO-d6), δ, ppm: 2.41 (s, 2H, CH3), 7.39–7.47 (m, 4H, arH), 7.76–7.82 (m, 2H, arH),
8.10 (d, 2H, arH, J = 8.2 Hz). 13C NMR spectrum (DMSO-d6), δ, ppm: 21.07 (CH3), arC:[119.55
(CH), 123.54 (2C), 124.72 (CH), 125.24 (CH), 127.13 (3CH), 129.81 (3CH), 142.08 (2C)], 141.40
(triazole C5), 149.99 (triazole C3). Anal. for: C15H13N3O Calcd. (%): C, 71.70, H, 5.21, N, 16.72.
Found, (%): C, 71.88, H, 5.27, N, 16.57.
4-[3-(1H-Imidazol-1-yl)propyl]-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (6). Recrystallized from
ethanol; yield 67%, m.p. 105–106 ºC. IR spectrum (KBr), ν, cm-1: 3106 (NH), 1696 (C=O), 1592

Molecules 2010, 15
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(C=N). 1H NMR spectrum (DMSO-d6), δ, ppm: 1.97 (t, 2H, CH2, J = 7.0 Hz), 2.06 (s, 3H, CH3), 3.47
(t, 2H, CH2, J = 7.0 Hz), 3.95 (t, 2H, CH2, J = 7.0 Hz), 6.87 (s, 1H, arH), 7.17 (s, 1H, arH), 7.65 (s,
1H, arH), 11.43 (s, 1H, NH). 13C NMR spectrum (DMSO-d6), δ, ppm: 15.93 (CH3), 34.73 (CH2),
42.09 (CH2), 48.03 (CH2), arC: [123.91 (CH), 133.00 (CH), 141.94 (CH)], 148.99 (triazole C3),
159.71 (triazole C5); MS (ESI): m/z (%) 230 (M+Na, 21), 208 (M+1, 15), 140 (100), 112 (22). Anal.
for: C9H13N5O Calcd. (%): C, 52.16, H, 6.32, N, 33.79. Found, (%): C, 52.28, H, 6.49, N, 33.57.
4-[2-(1H-Indol-3-yl)ethyl]-5-phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one (8). Recrystallized from
ethanol; yield 37%, m.p. 205–206 ºC. IR spectrum (KBr), ν, cm-1: 3397 (NH), 3379 (NH), 1708
1
(C=O), 1618 (C=N). H NMR spectrum (DMSO-d6), δ, ppm: 2.88 (t, 2H, CH2, J = 7.2 Hz), 3.90 (t,
2H, CH2, J = 7.2 Hz), 6.89 (t, 1H, arH, J = 7.8 Hz), 7.00-7.07 (m, 2H, arH), 7.19 (d, 1H, arH, J = 8.2
13
Hz), 7.30 (d, 1H, arH, 8.0 Hz), 7.42-7.52 (m, 6H, arH), 10.84 (s, 1H, NH), 11.93 (s, 1H, NH). C
NMR spectrum (DMSO-d6), δ, ppm: 24.05 (CH2), 41.81 (CH2), ar C:[109.83 (C), 111.24 (C), 117.63
(C), 118.21 (C), 120.85 (C), 122.87 (C), 126.76 (C), 127.31 (C), 127.80 (2C), 128.63 (2C), 129.84 (C),
136.01 (C)], 150.99 (triazole C3), 155.02 (triazole C5). MS (ESI): m/z (%) 327 (M+Na, 100), 324 (16),
305 (M+, 24), 189 (25), 188 (25), 153 (22), 148 (34), 144 (94), 121 (24). Anal. for: C18H16N4O Calcd.
(%): C, 71.04, H, 5.30, N, 18.41. Found, (%): 71, 18, H, 5.1, N, 18.27.
5-Methyl-4-(3-{4-[3-(3–methyl-5-oxo-1,5-dihydro-4H-1,2,4–triazol–4-yl)propyl]
piperazin-1-
yl}propyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (9a). Recrystallized from ethanol; yield 59%,
1
m.p. 246–248 ºC. IR spectrum (KBr), ν ,cm-1: 3177 (NH), 1707 (C=O), 1586 (C=N). H NMR
spectrum (DMSO-d6), δ, ppm: 1.74 (brs, 4H, 2CH2), 2.15-2.24 (brs, 18H, 6CH2+2CH3), 3.40 (brs, 4H,
2CH2), 11.31 (s, 2H, 2NH). 13C NMR spectrum (DMSO-d6), δ, ppm: 11.31 (CH3), 25.15 (2CH2),
52.54 (6CH2), 54.51 (2CH2), 144.51 (triazole C3), 154.96 (triazole C5). Anal. for: C16H18N8O2 Calcd.
(%): C, 49.99, H,7.19, N, 33.31. Found, (%): C, 50.18, H, 6.95, N, 33.17.
5-(4-Chlorobenzyl)-4-(3-{4-[3-(3-methyl-5-oxo-1,5-dihydro-4H-1,2,4-triazol-4-yl)propyl] piperazin-1-
yl}propyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (9b). Recrystallized from ethanol/ water [1:1]); yield
81%, m.p. 214–216 ºC. IR spectrum (KBr), ν ,cm-1: 3183 (NH), 1698 (C=O), 1569 (C=N).1H NMR
spectrum (DMSO-d6), δ, ppm: 1.51 (brs, 4H, 2CH2), 2.18 (brs, 12H, 6CH2), 3.43 (brs, 4H, 2CH2), 3.97
13
(brs, 4H, 2CH2), 7.30–7.39 (m, 8H, arH), 11.55 (brs, 2H, 2NH). C NMR spectrum (DMSO-d6), δ,
ppm: 24.82 (2CH2), 30.53 (2CH2), 52.44 (6CH2), 54.30 (2CH2), arC: [128.50 (4CH), 130.34 (4CH),
131.54 (2C), 134.55 (2C)], 145.97 (2 triazole C-5), 155.01 (2 triazole C-3). Anal. for: C28H34CI2N8O2
Calcd. (%): C, 56.02 H, 5.42 N, 20.10. Found, (%): C, 56.24 H, 5.80 N, 19.91.
2-[(4-Chlorophenyl)sulfonyl]-4-[2-(1H-indol-3-yl)ethyl]-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one
(7). Compound 6 (10 mmol) was refluxed with 1 equivalent of sodium in dichloromethane for 8 hours.
Then, 4-chlorobenzenesulphonyl chloride (10 mmol) was added and refluxed for an additional 8 hours.
After evaporating the solvent under reduced pressure, a solid appeared. This was recrystallized form
ethanol to afford compound 7. Yield 48%, m.p. 147–149 ºC. IR spectrum (KBr), ν, cm-1: 1730 (C=O),
1603 (C=N). 1H NMR spectrum (DMSO-d6), δ, ppm: 2.14 (t, 2H, CH2, J = 6.4 Hz), 2.21 (s, 3H, CH3),
3.53 (t, 2H, CH2, J = 6.6 Hz), 4.16 (t, 2H, CH2, J = 6.6 Hz), 7.38 (d, 1H, arH, J = 8.2 Hz), 7.60 (d, 2H,
arH, J = 8.6 Hz), 7.80–7.73 (m, 2H, arH), 7.95 (d, 1H, arH, J = 8.6 Hz), 8.95 (s, 1H, arH). 13C NMR

Molecules 2010, 15
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spectrum (DMSO-d6), δ, ppm: 11.51 (CH3), 28.29 (CH2), 36.86-40.58 (DMSO-d6 + CH2), 45.57
(CH2), arC: [125.74 (2CH), 127.54 (2CH), 128.38 (CH), 129.28 (CH), 129.94 (CH), 135.27 (C),
140.02 (C)], 148.86 (triazole C3), 150.95 (triazole C5). MS (ESI): m/z (%) 384 (M+2, 26), 382 (M+,
66), 316 (64), 314 (100), 175 (13). Anal. for: C15H16CIN5O3S Calcd. (%): C, 47.18 H, 4.22 N, 18.34.
Found, (%): C, 47.37, H, 4.33 N: 18.28.
Ethyl
[3-(4-chlorophenyl)-5-oxo-4-{[phenylmethylene]amino}-4,5-dihydro-1H-1,2,4-triazol-1-
yl]acetate (11). The corresponding compound 10 (10 mmol) was refluxed with 1 equivalent of sodium
in absolute ethanol for 2 hours. Then, ethyl bromoacetate (10 mmol) was added and refluxed for an
additional 8 hours. After evaporating the solvent under reduced pressure, a solid appeared. This was
recrystallized from ethanol/water (1:2) to afford compound 11. Yield 95%, m.p. 131–132 ºC. IR
1
spectrum (KBr), ν, cm-1: 1742 (C=O), 1714 (C=O), 1579 (C=N), 1230 (C-O). H NMR spectrum
(DMSO-d6), δ, ppm: 1.18–1.26 (t, 3H, CH3, J = 7.4 Hz ), 4.13-4.23 (q, 2H, CH2CH3, J = 7.4 Hz), 4.77
(s, 2H, NCH2), 7.48–7.65 (m, 5H, ar-H), 7.81–7.95 (m, 4H, ar-H), 9.62 (s, N=CH).13C NMR spectrum
(DMSO-d6), δ, ppm: 15.81 (CH3), 48.65 (CH2), 63.18 (CH2), arC: [126.51 (C), 129.91 (2C), 130.65
(2C), 130.92 (2C), 131.59 (C), 133.77 (C), 134.75 (C), 137.14 (C), 144.70 (C)], 144.7 (N=CH), 151.54
(triazole C-3), 159.24 (triazole C-5), 169.28 (C=O). MS (ESI): m/z (%) 385.82 (M+1, 26), 272 (80).
Anal. Calcd. (%) for: C19H17N4CIO3: C, 59.30, H, 4.45, N, 14.56. Found, (%): C, 59.20, H, 4.42,
N,14.55.
2-[4-amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]aceto hydrazide (12).
A
solution of the corresponding compound 11 (10 mmol) in n-butanol was refluxed with hydrazine
hydrate (25 mmol) for 4 hours. After cooling it to room temperature, a white solid appeared. This was
recrystallized from ethanol to afford the desired product. Yield 98%, m.p. 215–218 ºC. IR spectrum
1
(KBr), ν, cm-1: 3302–3213 (NH+2NH2), 1717 (C=O), 1668 (C=O), 1577 (C=N). H NMR spectrum
(DMSO-d6), δ, ppm: 4.34 (s, 2H, NCH2), 4.46 (s, 2H, NHNH2), 5.55 (s, 2H, NH2), 7.52-7.65 (m, 2H,
ar-H), 7.82-8.05 (m, 2H, ar-H), 9.27 (s, NHNH2). 13C NMR spectrum (DMSO-d6), δ, ppm: 48.69
(CH2), arC: [126.82 (C), 127.38 (C), 133.82 (C), 134.93 (C), 138.47(C), 138.97 (C)], 151.79 (triazole
C-3), 158.55 (triazole C-5), 167.45 (C=O). MS (ESI): m/z (%) 283.69 (M+1, 32), 305 (100), 273 (34),
229(48). Anal. Calcd. (%) for: C10H11N6ClO2: C, 42.49, H, 3.92, N, 29.73. Found, (%): C, 42.50, H,
3.90, N,29.65.
4-Amino-5-(4-chlorophenyl)-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-2,4-dihydro-3H-1,2,4-
triazol-3-one (13). Compound 12 (10 mmol) and CS2 (6.0 mL, 10 mol) were added to a solution of
KOH (0.56 g, 10 mol) in 50 mL H2O and 50 mL ethanol. The reaction mixture was refluxed for 3 h.
After evaporating in reduced pressure to dryness, a solid was obtained. This was dissolved in 300 mL
H2O and acidified with conc. HCl. The precipitate was filtered off, washed with H2O and recrystallized
from ethanol to afford the desired compound.Yield 89%, m.p. 220–221 ºC. IR spectrum (KBr), ν, cm-1:
3323–3203 (NH2), 2750 (SH), 1679(C=O), 1518, 1492 (C=N), 1164 (C=S).1H NMR (DMSO-d6), δ,
ppm: 5.18 (s, 2H, CH2), 5.63 (bs, 2H, NH2), 7.55–7.59 (d, 2H, arH, J = 8.4 Hz), 7.98–8.02 (d, 2H,
arH, J = 8.4 Hz), 14.65 (bs, 1H, SH).13C NMR spectrum (DMSO-d6), δ, ppm: 38.085–40.172 (DMSO-
d6 + CH2), arC: [125.14 (2C), 128.45 (C), 130.80 (2C), 134.12 (C)], 152.70 (triazole C-3), 155.12
(triazole C-5), 160.12 (oxadiazole C-2), 174.12 (oxadiazole C-5). MS (ESI): m/z (%) 363 (100), 347

Molecules 2010, 15
2434
(M+Na, 12), 325.74 (M+1, 36), 223 (54), 123 (84). Anal. Calcd. (%) for: C11H9N6ClO2S: C, 40.86, H,
2.79, N, 25.88. Found, (%): C, 40.62, H, 2.80, N,25.80.
5-(4-Chlorophenyl)-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-4-[(4-methoxyben-zylidene)amino]-
2,4-dihydro-3H-1,2,4-triazol-3-one (14). The mixture of compound 13 (10 mmol) and anisaldehyde
(10 mmol) in absolute ethanol was refluxed for 5h. On cooling it to room temperature, a solid
appeared. This crude product was recryristallized from ethanol: water (1:1) to obtain compound 14.
Yield 71%, m.p. 229–230 ºC. IR spectrum (KBr), ν, cm-1: 2747 (SH), 1705 (C=O), 1628, 1605, 1566
(3C=N), 1170 (C=S). 1H NMR spectrum (DMSO-d6), δ, ppm: 3.84 (s, 3H, OCH3), 5.28 (s, 2H, NCH2),
7.05–7.09 (d, 2H, ar-H, J = 8.6 Hz), 7.58–7.62 (d, 2H, ar-H, J = 8.2 Hz), 7.66–7.81(d, 2H, ar-H,
J =8.6 Hz), 7.90-7.94 (d, 2H, ar-H, J =8.2 Hz), 9.49 and 9.63 (s, 1H, -N=CH, E/Z geometrical
13
izomers), 14.74 (bs, 1H, SH). C NMR spectrum (DMSO-d6), δ, ppm: 38.085-40.17 (DMSO-d6 +
CH2), 55.31 (OCH3), arC: [114.43 (2C), 124.35 (C), 125.15 (2C), 127.96 (C), 128.66 (2C), 129.86
(2C), 135.24 (C), 162.19 (C)], 143.21 (triazole C-3), 149.21 (triazole C-5), 157.73 (C=N), 158.47
(oxadiazole C-2), 177.96 (oxadiazole C-5). MS (ESI): m/z (%) 443.85 (M+1) (42), 305 (80), 131 (56),
123 (73). Anal. Calcd. (%) for: C19H15N6ClO3S: C, 51.53, H, 3.41, N, 18.98. Found, (%): C, 51.48, H,
3.45, N,18.90.
3.3. General Method for the synthesis of compounds 15a, 15b
To the solution of corresponding compound 14 (10 mmol) in dichloromethane, formaldehyde (37%,
1.55 mL) and methyl piperazine (for 15a) or morpholine (for 15b) (10 mmol) were added and the
mixture was stirred at room temperature for 3 h. After removing the solvent under reduce pressure, a
solid was obtained. This crude product was treated with water, filtered off and recrystallized from
ethyl acetate/petroleum ether (1:2) to yield the title compounds.
4-[(4-Methoxybenzylidene)amino]-5-(4-chlorophenyl)-2-({4-[(4-methylpiperazin-1-yl)methyl]-5-
thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl}methyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (15a). Yield 75%,
m.p. 165–167 ºC. IR spectrum (KBr), ν, cm-1: 2985 (CH), 1715 (C=O), 1616,1510 (C=N), 1330 (C=S).
1H NMR spectrum (DMSO-d6), δ, ppm: 2.29 (s, 3H, NCH3), 2.52 (bs, 4H, 2CH2), 2.78 (bs, 4H, 2CH2),
3.84 (s, 3H, OCH3), 4.98 (s, 2H, CH2), 5.21 (s,2H, NCH2), 7.06–7.10 (d, 2H, ar-H, J = 8.6 Hz),
7.60–7.64 (d, 2H, ar-H, j =8.2 Hz), 7.78–7.83 (d, 2H, ar-H, J = 8.6 Hz), 7.91–7.95 (d, 2H, ar-H,
J = 8.2 Hz), 9.50,9.64 (s, 1H, -N=CH, E/Z geometrical izomers). 13C NMR (DMSO-d6), δ, ppm: 44.58
(NCH3), 48.68 (2C, 2CH2), 52.30 (2C, 2CH2), 53.65 (NCH2), 55.38 (OCH3), 69.39 (NCH2N), arC:
[114.52 (2C), 124.58 (C), 125.22 (C), 128.71 (2C), 129.04 (2C), 129.95 (2C), 135.21 (C), 162.24 (C)],
143.10 (triazole C-3), 149.37 (triazole C-5), 156.97 (C=N), 157.88 (oxadiazole C-2), 164.22
(oxadiazole C-5). Anal. Calcd. (%) for: C25H27N8ClO3S: C, 54.10, H, 4.90, N, 20.19. Found, (%): C,
54.11, H, 4.88, N, 20.15.
4-[(4-Methoxybenzylidene)amino]-5-(4-chlorophenyl)-2-{[4-(morpholin-4-ylmethyl)-5-thioxo-4,5-
dihydro-1,3,4-oxadiazol-2-yl}methyl)-2,4-dihydro-3H-1,2,4-triazol-3-one
(15b).
Yield
78%,
m.p. 175–176 ºC. IR spectrum (KBr), ν, cm1: 1712 (C=O), 1606, 1515 (2C=N), 1327 (C=S). 1HNMR
spectrum (DMSO-d6), δ, ppm: 2.69 (t, 4H, 2CH2), 3.56 (t, 4H, 2CH2), 3.84 (s, 3H, OCH3), 4.97 (s, 2H,

Molecules 2010, 15
2435
CH2), 5.30 (s, 2H, NCH2), 7.05–7.10 (d, 2H, ar-H, J =8.8 Hz), 7.60-7.64 (d, 2H, ar-H, J = 8.6 Hz),
7.78–7.82 (d, 2H, ar-H, J = 8.6 Hz), 7.90–7.94 (d, 2H, ar-H, J = 8.8 Hz), 9.49, 9.63 (s,1H, N=CH, E/Z
geometrical izomers).13CNMR spectrum (DMSO-d6), δ, ppm: 50.5 (2C, 2CH2), 56.137 (OCH3), 64.16
(NCH2), 66.65 (2C, 2CH2), 70.43 (NCH2N), arC: [115.28 (2C), 125.29 (C), 125.95 (C), 129.48 (2C),
130.49 (2C), 130.72 (2C), 136.07 (C), 163.03 (C)], 144.08 (triazole C-3), 150.18 (triazole C-5), 157.61
(C=N), 158.55 (oxadiazole C-2), 178.68 (oxadiazole C-5). MS (ESI): m/z (%) 542 (M+, 20), 508 (32),
292 (30), 215 (83), 210 (94), 153 (100). Anal. Calcd. (%) for: C24H24N7ClO4S: C,53.18, H, 4.46, N,
18.09. Found, (%): C, 53.15, H, 4.45, N,18.12.
3.4. Antimicrobial activity assessment
All test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam (Ankara,
Turkey) and were as follows: Escherichia coli ATCC 35218, Klebsiella pneumoniae ATCC 13883,
Yersinia pseudotuberculosis ATCC 911, Enterobacter aerogenes ATCC 13048, Pseudomonas
aeruginosa ATCC 10145, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212,
Bacillus cereus 709 Roma, Candida tropicalis ATCC 13803, Candida glabrata 66032 and Candida
albicans ATCC 60193. All the newly synthesized compounds were weighed and dissolved in
dimethylsulphoxide to prepare extract stock solution of 10.000 microgram/milliliter (μg/mL).
The antimicrobial effects of the substances were tested quantitatively in respective broth media by
using double dilution and the minimal inhibition concentration (MIC) values (µg/mL) were determined
[35,36]. The antibacterial and antifungal assays were performed in Mueller-Hinton broth (MH) (Difco,
Detroit, MI) at pH 7.3 and buffered Yeast Nitrogen Base (Difco, Detroit, MI) at pH 7.0, respectively.
The MIC was defined as the lowest concentration that showed no growth. Ampicillin (10 μg) was used
as standard antibacterial and antifungal drugs, respectively. Dimethylsulphoxide with dilution of 1:10
was used as solvent control. The results are shown in Table 1.
Table 1. Antimicrobial activity of the synthesized compounds (µg/mL).
Compound No.
Microorganisms and inhibition zone (mm)
Ec
250
Kp
250
>500
>500
>500
250
>500
>500
>500
>500
<1.95
<1.95
500
Yp
En
Pa
Sa
Ef
Bc
Ca
Ct
3
5
6
7
8
9a
9b
11
12
13
>500
>500
>500
>500
>500
>500
>500
>500
>500
<1.95
<1.95
250
>500
>500
>500
>500
>500
>500
>500
>250
62.5
<1.95
<1.95
7.81
<1.95
>128
>500
>500
>500
>500
>500
>500
>500
>500
>500
<1.95
<1.95
250
>500
>500
>500
>500
>500
>500
>500
250
>500
>500
>500
>500
>500
>500
>500
250
>500
>500
>500
>500
>500
>500
>500
>250
62.5
<1.95 >500
<1.95 >500
1.95
<1.95 >500
15
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
8
>500
>500
>500
250
>500
>500
>500
>500
<1.95
<1.95
>500
<1.95
10
125
125
<1.95
<1.95
15.63
<1.95
35
<1.95
<1.95
125
<1.95
10
14
15a
15 b
Amp.
Flu.
>500
<1.95
>128
<1.95
18
<1.95
18
>500
<1
Ec: Escherichia coli ATCC 25922, Yp: Yersinia pseudotuberculosis ATCC 911, Pa: Pseudomonas
aeruginosa ATCC 27853, Ef: Enterococcus faecalis ATCC 29212, Sa: Staphylococcus aureus
ATCC 25923, Kp: Klebsiella pneumoniae ATCC 13883, En: Enterobacter aerogenes ATCC 13048;
Bc: Bacillus cereus 702 Roma, Ct: Candida tropicalis ATCC 13803, Ca: Candida albicans ATCC
60193, Amp: Ampicillin, Flu: Fluconazole.

Molecules 2010, 15
2436
4. Conclusion
This study reports the successful synthesis of some new 1,2,4-triazol-3-one derivatives, one of them
into the corresponding Shiff and Mannich bases. The antimicrobial screening studies were also
performed in the study. 1,2,4-Triazole nucleus is one of the active components present in many
standard drugs and it is known to increase the pharmacological activity of the molecules. The presence
of N-methylpiperazine or morpholine moiety is also instrumental in contributing to the net biological
activity of a system.
Also we already reported antimicrobial activities of some biheterocyclic triazole derivatives
incorporating indole, imidazole, 1,3,4-oxadiazole and piperazine moieties. Hence herein we combined
all these potential units, namely 1,2,4-triazole and 1,3,4-oxadiazole, imidazole, indole, morpholine,
piperazine or methyl piperazine ring.
The antimicrobial screening suggests that among the newly synthesized compounds, the compounds
3 and 8 exhibited moderate activities towards Escherichia coli (Ec) and Klebsiella pneumoniae (Kp),
similarly, compounds 11 and 12 showed moderate activities against Enterobacter aerogenes (En),
Staphylococcus aureus (Sa), Enterococcus faecalis (Ef) and Bacillus careus (Be); while good
antimicrobial activities were found for compounds 13 and 14 against the test microorganisms. Also the
Mannich bases 15a,b displayed good or moderate antimicrobial activities against the test
microorganisms. On the other hand, none of the synthesized compounds showed antimicrobial activity
against Candida tropicalis (Ct) and Candida albicans (Ca).
Acknowledgements
This work was supported by the Scientific and Technological Research Council of Turkey
(TUBITAK, Project no: 107T333).
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Sample Availability: Samples of the compounds are available from authors.
© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).