Synthesis, in vitro antimicrobial and antioxidant activities of some new 4,5-dihydro-1h-1,2,4-triazol-5-one derivatives

Article abstract of DOI:10.1002/ardp.201300048

A series of compounds derived from 4,5-dihydro-1H-1,2,4-triazol-5-one were synthesized and characterized by spectral data. The 12 new compounds were analyzed for their potential in vitro antioxidant activities by three different methods. Compound 4f showe

Full text of DOI:10.1002/ardp.201300048

Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
1
Full Paper
Synthesis, In Vitro Antimicrobial and Antioxidant Activities
of Some New 4,5-Dihydro-1H-1,2,4-triazol-5-one Derivatives
1
2
3,4
1
3
¨
Haydar Y u¨ ksek , Onur Akyıldırım , Mehmet L. Yola , Ozlem G u¨ rsoy-Kol , Mustafa C¸ elebier ,
5
and Didem Kart
1
Department of Chemistry, Faculty of Science and Letters, Kafkas University, Kars, Turkey
Department of Chemical Engineering, Faculty of Engineering and Architecture, Kafkas University, Kars,
2
Turkey
Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye, Ankara, Turkey
Department of Chemistry, Faculty of Science and Letters, Sinop University, Sinop, Turkey
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Hacettepe University, Sıhhiye, Ankara,
Turkey
3
4
5
A series of compounds derived from 4,5-dihydro-1H-1,2,4-triazol-5-one were synthesized and
characterized by spectral data. The 12 new compounds were analyzed for their potential in vitro
antioxidant activities by three different methods. Compound 4f showed the best activity for the iron
binding. In addition, the compounds 4 were titrated potentiometrically with tetrabutylammonium
0
hydroxide in non-aqueous solvents. The RP-HPLC capacity factors (k ) of the series were also determined
0
on a C18 column, with methanol/water as the mobile phase. The correlation between log k with the
percentage of methanol in the mobile phase was used for the determination of the log k values for
w
these compounds. The antimicrobial activities of these compounds were also screened against
bacteria and yeast.
Keywords: 1,2,4-Triazol-5-one / Acidity / Antimicrobial activity / Antioxidant activity / log k
Received: February 8, 2013; Revised: March 21, 2013; Accepted: March 28, 2013
DOI 10.1002/ardp.201300048
w
Introduction
as cancer, emphysema, cirrhosis, atherosclerosis, and
arthritis which have all been correlated with oxidative
damage. Also, the excessive generation of ROS induced
by various stimuli which exceeds the antioxidant capacity
of the organism leads to variety of pathophysiological
processes such as inflammation, diabetes, genotoxicity,
and cancer [2].
Antioxidants have extensively been studied for their capacity
to protect organisms and cells from damages that are
induced by oxidative stress. Scientists have become more
interested in new compounds; they have either synthesized
or obtained them from natural sources that could provide
active components for preventing or reducing the impact
of oxidative stress on cells [1]. Exogenous chemicals and
endogenous metabolic processes in human body or in
food system might produce highly reactive free radicals,
especially oxygen, derived from radicals which are
capable of oxidizing biomolecules that result in the cell
death and tissue damage. Oxidative damages significantly
play a pathological role in serious human diseases such
It is a well-known fact that there is a relationship between
the affinity to lipids and biological activity of the chemical
substances, which is generally defined as the tendency of a
chemical to distribute between an immiscible nonpolar sol-
vent and water. Lipophilicity or lipid/water partition proper-
ties affect most of the processes at the basis of drug action [3,
a
4]. The acid–base dissociation constant (pK ) of a drug is
another important physicochemical parameter influencing
many biopharmaceutical characteristics. In the case of ion-
izable drugs, the process in the pharmacokinetic phase of
drug action (absorption, distribution, and excretion) depend
additionally on the dissociation of the drug in aqueous com-
partments of a living system separated by lipid membranes
¨
Correspondence: Ozlem G u¨ rsoy-Kol, Department of Chemistry, Faculty
of Science and Letters, Kafkas University, 36100 Kars, Turkey.
E-mail: ozlemgursoy@gmail.com
Fax: þ90 4742251179
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
H. Y u¨ ksek et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
[
5]. Therefore, determining lipophilicity (hydrophobicity)
dihydro-1H-1,2,4-triazol-5-ones 3a–g with 3-methoxy-4-phe-
nylacetoxybenzaldehyde 1 which were synthesized by the
reactions of 3-methoxy-4-hydroxybenzaldehyde with phenyl-
acetyl chloride by using triethylamine. Then, the reactions of
compounds 4a, 4d, 4e, 4f, and 4g with acetic anhydride were
investigated, and compounds 5a, 5d, 5e, 5f, and 5g were
prepared (Scheme 1).
parameters and ionization constants of drug candidates is
necessary at the early stages of the drug development process
[6, 7]. Application of chromatographic retention in quanti-
tative structure property/activity relationship studies is an
active research field, which attracts many analytical chem-
istry, environmental chemistry, and pharmaceutical chem-
istry researchers. The logarithm of the partition coefficient of
a chemical in the n-octanol/water system (kow), which is
usually measured by the ‘‘shake flask’’ method, is widely
used because of its simplicity and some similarity between
n-octanol and biological membranes. However, the conven-
tional ‘‘shake flask’’ method has a limited application range
up to log kow ¼ 4, and it is time consuming and also requires
considerable amounts of pure stable compounds. It has been
The structures of seven new 3-alkyl(aryl)-4-(3-methoxy-
4-phenylacetoxybenzylidenamino)-4,5-dihydro-1H-1,2,4-tri-
azol-5-one 4a–g compounds, and five new 1-acetyl-3-alkyl(aryl)-
4-(3-methoxy-4-phenylacetoxybenzylidenamino)-4,5-dihydro-
1H-1,2,4-triazol-5-one 5a, 5d, 5e, 5f, and 5g compounds were
1
13
identified by using IR, H NMR, C NMR, UV, and elemental
analysis data.
0
proven that the retention capacity factor (k ) of a compound
Antioxidant activity
in a reversed-phase high performance liquid chromatog-
raphy (RP-HPLC) system is a reliable indirect descriptor of
the lipophilicity of a compound [8, 9].
The antioxidant activities of 12 new 4a–g, 5a, 5d, 5e, 5f, and
5g compounds were determined. Several methods have been
used to determine antioxidant activities and the methods
used in the study are given below.
1
,2,4-Triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one deriva-
tives have been found to have a broad spectrum of biological
activities [10–18]. In addition, several articles about the syn-
thesis of some N-arylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-
one derivatives have been published [17–20]. The acetylation
of 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives has also been
reported [17–23].
Total reductive capability using the potassium ferricyanide
reduction method
The reductive capabilities of compounds were assessed by the
3þ
extent of conversion of the Fe /ferricyanide complex to the
2
þ
Fe /ferrous form. The reducing powers of the compounds
were observed at different concentrations, and results were
compared with BHA, BHT, and a-tocopherol (standard anti-
oxidants). It has been observed that the reducing capacity of a
compound may serve as a significant indicator of its potential
antioxidant activity [27]. The antioxidant activity of putative
antioxidant has been attributed to various mechanisms such
as prevention of chain initiation, binding of transition metal
ion catalyst, decomposition of peroxides, prevention of con-
tinued hydrogen abstraction, reductive capacity, and radical
scavenging [28]. In the study, all the amount of the com-
pounds showed lower absorbance when compared to the
blank. Hence, no activities were observed to reduce metal
ions complexes to their lower oxidation state or to take part
in any electron transfer reaction. In other words, the com-
pounds did not show the reductive activities.
In the present study, due to a wide range of applications to
find their possible radical scavenging and antioxidant
activity, the newly synthesized compounds were investigated
by using different antioxidant methodologies: 1,1-diphenyl-2-
ꢀ
picryl-hydrazyl (DPPH ) free radical scavenging, reducing
power and metal chelating activities. Besides, it is known
that 1,2,4-triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one rings
have weak acidic properties, so that some 1,2,4-triazole and
4
,5-dihydro-1H-1,2,4-triazol-5-one derivatives were titrated
potentiometrically with tetrabutylammonium hydroxide
TBAH) in non-aqueous solvents, and the pK values of
the compounds were determined [17–22, 24, 25]. The
,2,4-triazole compounds have been known to possess anti-
(
a
1
microbial activities, especially antifungal activity. The anti-
microbial evaluation of 4,5-dihydro-1H-1,2,4-triazol-5-one
derivatives was also reported as potential antimicrobial
agents [26]. According to these results, antimicrobial activi-
ties of some 1,2,4-triazole and 4,5-dihydro-1H-1,2,4-triazol-5-
one derivatives were screened.
ꢀ
DPPH radical scavenging activity
The scavenging of stable DPPH radical model is a widely used
method to evaluate antioxidant activities in a relatively short
time in comparison with other methods. The effect of anti-
oxidants on DPPH radical scavenging was thought to be due
to their hydrogen donating ability [29]. DPPH is a stable free
radical and accepts an electron or hydrogen radical to
become a stable diamagnetic molecule [30]. The reduction
capability of DPPH radicals was determined by decrease in
its absorbance at 517 nm induced by antioxidants. The
Results and discussion
Chemistry
The 3-alkyl(aryl)-4-(3-methoxy-4-phenylacetoxybenzylidena-
mino)-4,5-dihydro-1H-1,2,4-triazol-5-ones 4a–g were obtained
from the reactions of compounds 3-alkyl(aryl)-4-amino-4,5-
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
4,5-Dihydro-1H-1,2,4-triazol-5-one Derivatives
3
Scheme 1. Synthesis route of compounds 1, 3–5.
3
living organism. The ferric iron (Fe ) is a relatively biological
þ
absorption maximum of a stable DPPH radical in ethanol was
at 517 nm. The decrease in absorbance of the DPPH radical
was caused by antioxidants, because of reaction between
antioxidant molecules and radical progresses, which resulted
in the scavenging of the radical by hydrogen donation. It is
visually noticeable as a discoloration from purple to yellow.
inactive form of iron. However, it can be reduced to the active
2
þ
Fe , depending on condition, particularly pH [33] and oxi-
dized back through Fenton type reactions with the pro-
duction of hydroxyl radical or Haber–Weiss reactions with
superoxide anions. The production of these radicals may lead
to lipid peroxidation, protein modification, and DNA dam-
age. Chelating agents may not activate metal ions and poten-
tially inhibit the metal-dependent processes [34]. Also, the
ꢀ
Hence DPPH is usually used as a substrate to evaluate the
antioxidative activity of antioxidants [31]. In the study, anti-
radical activities of compounds and standard antioxidants
such as BHA and a-tocopherol were determined by using the
ꢀ
ꢁ
ꢀ
production of highly active ROS such as O2 , H O , and OH
2
2
ꢀ
DPPH method. The newly synthesized compounds showed
are also catalyzed by free iron though Haber–Weiss reactions:
no activity as a radical scavenger.
ꢀ
ꢁ
ꢀ
O2 þ H2O2 ! O2 þ OH þ OH
Ferrous ion chelating activity
The chelating effect towards ferrous ions by the compounds
Among the transition metals, iron is known as the most
important lipid oxidation pro-oxidant due to its high reac-
tivity. The ferrous state of iron accelerates lipid oxidation by
breaking down the hydrogen and lipid peroxides to reactive
free radicals via the Fenton reactions:
and standards was determined. Ferrous ion can form quan-
2
þ
titatively complexes with Fe . In the presence of chelating
agents, the complex formation is disrupted with the result
that the red color of the complex is decreased. Measurement
of color reduction, therefore, allows estimation of the chelat-
ing activity of the coexisting chelator [32]. Transition metals
have pivotal role in the generation of oxygen free radicals in
Fe þ H2O2 ! Fe3þ þ OH þ OH
2
þ
ꢁ
ꢀ
3
Fe ion also produces radicals from peroxides, even though
þ
2
the rate is 10-fold less than that of Fe ion, which is the most
þ
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4
H. Y u¨ ksek et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
Figure 1. Metal chelating effect of different amounts of the compounds 4a–g, BHT, BHA, and a-tocopherol on ferrous ions.
powerful pro-oxidant among the various types of metal ions
35]. Ferrous ion chelating activities of the compounds 4, 5,
their action as peroxidation protectors may be related to
their iron binding capacity. On the other hand, free iron is
known to have low solubility and a chelated iron complex has
greater solubility in solution, which can be contributed
solely by the ligand. Furthermore, the compound–iron
complex may also be active, since it can participate in
iron-catalyzed reactions.
[
BHT, BHA, and a-tocopherol are respectively shown in Figs. 1
and 2.
In this study, metal chelating capacity was significant since
it reduced the concentrations of the catalyzing transition
metal. It was reported that chelating agents that form s-
bonds with a metal are effective as secondary antioxidants
because they reduce the redox potential thereby stabilizing
the oxidized form of metal ion [36]. The data obtained from
Figs. 1 and 2 reveal that the compounds, especially 4f, dem-
onstrates a marked capacity for iron binding, suggesting that
Potentiometric titrations
a
In order to determine the pK values of the compounds 4a–g,
they were titrated potentiometrically with TBAH in four
non-aqueous solvents: isopropyl alcohol, tert-butyl alcohol,
Figure 2. Metal chelating effect of different amounts of the compounds 5a, 5d, 5e, 5f and 5g, BHT, BHA, and a-tocopherol on ferrous ions.
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
4,5-Dihydro-1H-1,2,4-triazol-5-one Derivatives
5
acetone and DMF. The mV values read in each titration were
plotted against 0.05 M TBAH volumes (mL) added, and poten-
tiometric titration curves were obtained for all the cases.
From the titration curves, the HNP values were measured,
alcohol > acetone, for compound 4d it is: isopropyl
alcohol > DMF > tert-butyl alcohol, for compound 4f it is:
tert-butyl alcohol > DMF, while the order for compound 4g is:
acetone > tert-butyl alcohol > DMF. Moreover, as seen in
Table 1, for compounds 4c, 4e, 4f, and 4g in isopropyl alcohol,
for compound 4a and 4c in DMF, for compounds 4a and 4e in
tert-butyl alcohol and for compounds 4b, 4d, 4e, and 4f in
a
and the corresponding pK values were calculated. The data
obtained from the potentiometric titrations was interpreted,
and the effect of the C-3 substituent in 4,5-dihydro-1H-1,2,4-
triazol-5-one ring as well as solvent effects were studied [18,
acetone, the HNP values and the corresponding pK values
a
1
9, 21, 22, 24, 25]. Determination of pK values of the active
a
were not obtained.
constituent of certain pharmaceutical preparations is crucial
because the distribution, transport behavior, bonding to
receptors, and contributions to the metabolic behavior of
the active constituent molecules depend on the ionization
constant [37–39].
It is well known that the acidity of a compound depends on
some factors, of which the most two important are the
solvent effect and molecular structure [17–22, 24, 25, 40].
Table 1 and Fig. 3 show that the HNP values and
a
corresponding pK values obtained from the potentiometric
As an example for the potentiometric titration curves for
titrations depend on the non-aqueous solvents used and the
substituents at C-3, in the 4,5-dihydro-1H-1,2,4-triazol-5-one
ring.
0
.001 M solutions of compound 4g titrated with 0.05 M TBAH
in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone are
shown in Fig. 3.
While the dielectric permittivity of solvents is taken
into consideration, the acidity order can be as follows:
DMF (e ¼ 36.7) > acetone (e ¼ 36) > isopropyl alcohol
Calculation of log k values
w
0
The relationship between log k and methanol concentration
in mobile phase is known in HPLC theory [41], and it is
0
0
(
e ¼ 19.4) > tert-butyl alcohol (e ¼ 12). As seen in Table 1,
described with the formula: log k ¼ log k
w
ꢁ Sw where k
w
0
the acidity order for compound 4a is: acetone > isopropyl
alcohol, for compound 4b it is: tert-butyl alcohol >
DMF > isopropyl alcohol, for compound 4c it is tert-butyl
represents the k value for a compound if the pure water is
used as eluent, S is the slope of the regression curve, and w is the
volume percentage of methanol in the mobile phase. For each
Figure 3. Potentiometric titration curves of 0.001 M solutions of compound 4g titrated with 0.05 M TBAH in isopropyl alcohol, tert-butyl
alcohol, DMF, and acetone at 258C.
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6
H. Y u¨ ksek et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
Table 1. The HNP and the corresponding pK
Compound Isopropyl alcohol
HNP (mV) pK
17.20
a
values of compounds 4a–g in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone.
tert-Butyl alcohol
HNP (mV) pK
DMF
HNP (mV)
Acetone
HNP (mV) pK
a
a
a
pK
a
4a
4b
4c
4
4
4
4
ꢁ499
–
–
–
–
ꢁ290
11.82
ꢁ458
16.58
–
13.56
–
–
–
ꢁ266
ꢁ178
ꢁ671
–
12.15
10.10
–
ꢁ366
13.21
–
16.71
12.86
14.04
18.94
ꢁ
ꢁ
14.65
–
–
–
ꢁ537
d
e
f
ꢁ333
ꢁ523
ꢁ367
ꢁ371
ꢁ558
–
–
–
–
–
–
–
–
8.34
ꢁ305
13.45
11.55
–
g
ꢁ252
ꢁ147
studied compound, the linear correlation was found between
0
could prevent redox cycling. Design and synthesis of novel
small molecules can play specifically a protective role in
biological systems and in modern medicinal chemistry.
The results on the investigation of their biological activities
might be helpful in the future drug development process.
The synthesized compounds in this study were evaluated
according to their antioxidant, antibacterial, and antifungal
activity, and their lipophilicities were enlightened by finding
log k and w, and the correlation coefficients were all >0.99.
0
The values of S and the extrapolated log k are given in Table 2.
Figure 4 is given as an example to clarify this application.
Antimicrobial activity results
The synthesized compounds and their antibacterial and anti-
fungal activities are given in Table 3. The screening data
indicate that compound 4d shows the best antibacterial
activity against E. faecalis among the synthesized compounds.
All the studied compounds were found to be more effective
against E. faecalis and C. krusei than the other tested bacteria
and yeast strains, respectively.
w
log k values. These results may provide some guidance for
the development of novel triazole-based therapeutic targets.
Experimental
Chemical reagents and apparatus
Conclusion
Chemical reagents and all solvents in the study were purchased
from E. Merck (Darmstadt, Germany) and Sigma (Sigma–Aldrich
GmbH, Steinheim, Germany). The starting materials 3a–g were
prepared from the reactions of the corresponding ester ethoxy-
carbonylhydrazones 2a–g with an aqueous solution of hydrazine
hydrate as described in the literatures [23, 42]. Melting points
which were uncorrected were determined in open glass capil-
laries by using an Electrothermal digital melting point appar-
atus (Electrothermal Engineering Ltd., UK). The IR spectra were
obtained by a Perkin–Elmer Instruments Spectrum One FT-IR
spectrometer (potassium bromide disks; Perkin Elmer, UK).
The synthesis and in vitro antioxidant evaluation of new 4,5-
dihydro-1H-1,2,4-triazol-5-one derivatives are described in the
study. All of the compounds demonstrate a marked capacity
for the iron binding. The data reported with regard to the
observed metal chelating activities of the studied compounds
Table 2. Measured log k
w
values for the synthesized compounds.
1
13
H and C NMR spectra were recorded in deuterated dimethyl
sulfoxide with TMS as internal standard using a Varian Mercury
Compound
Measured values
1
200 NMR spectrometer (Varian Inc., UK) at 200 MHz for H and
wꢂꢂ
ꢂ
log k
S
1
3
0 MHz for C. UV absorption spectra were measured in 10 mm
5
4
4
4
4
4
4
4
5
5
5
5
5
a
b
c
d
e
f
g
a
d
e
f
3.499
4.021
4.527
4.777
5.336
5.518
4.673
3.920
5.266
4.719
4.771
5.127
0.043
0.048
0.052
0.055
0.060
0.062
0.054
0.047
0.059
0.052
0.053
0.058
quartz cells between 200 and 400 nm using a Shimadzu-1201 UV/
VIS spectrometer (Shimadzu, Tokyo, Japan). Extinction coeffi-
cients (e) are expressed in L mol cm . Elemental analyses
were carried out on a Leco 932 Elemental Combustion System
(CHNS-O) (Leco, Philadelphia, PA, USA) for C, H, and N.
ꢁ1
ꢁ1
General procedure for the synthesis of compounds 4
3-Methoxy-4-hydroxybenzaldehyde (0.01 mol) dissolved in ethyl
acetate (20 mL) was treated with phenylacetyl chloride
(0.01 mol) and to this solution was slowly added triethylamine
(0.01 mol) with stirring at 0–58C. The process of stirring contin-
ued for 2 h, and then the mixture was refluxed for 3 h and
filtered. The filtrate evaporated in vacuo, and the crude product
was washed with water and recrystallized from ethyl acetate–
g
ꢂ
S is the slope.
ꢂꢂ
0
log k
percentages are of methanol in the mobile phase).
w
is the intercept of the plot of log k versus w (the volume
petroleum ether to afford compound 1, mp 458C; UV (C
2 5
H OH)
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
4,5-Dihydro-1H-1,2,4-triazol-5-one Derivatives
7
Figure 4. Overlapped chromatograms of 4g obtained by using different mobile phases (90:10, 85:15, 80:20, 75:25 MeOH/water (v/v)).
l
max (log e) 302 (2.923), 258 (7.176), 220 (13.183), 206 (11.170) nm.
Ar-H), 9.70 (s, 1H, N–CH), 11.85 (s, 1H, NH); UV (C
(log e) 308 (11794), 264 (8725), 220 (14542), 216 (14504) nm; Anal.
calcd. for C19 (366.38): C, 62.28; H, 4.95; N, 15.29. Found:
C, 61.42; H, 4.93; N, 14.86.
2 5
H OH) lmax
The corresponding compound 3 (0.01 mol) was dissolved in
acetic acid (20 mL) and treated with 3-methoxy-4-phenylacetox-
ybenzaldehyde 1 (0.01 mol). The mixture was refluxed for 2 h
and then evaporated at 50–558C in vacuo. Several recrystalliza-
tions of the residue from ethanol gave pure compounds 3-alkyl-
18 4 4
H N O
3-Ethyl-4-(3-methoxy-4-phenylacetoxybenzylidenamino)-
(aryl)-4-(3-methoxy-4-phenylacetoxybenzylidenamino)-4,5-dihydro-
4,5-dihydro-1H-1,2,4-triazol-5-one 4b
1H-1,2,4-triazol-5-one 4 as colorless crystals.
Yield 2.88 g (75.80%). m.p. 1218C. IR (KBr) n 3192 (NH), 1762, 1702
ꢁ
1
(
C–O), 1598 (C–N), 1272 (COO), 755 and 700 cm (monosubsti-
3
-Methyl-4-(3-methoxy-4-phenylacetoxybenzylidenamino)-
,5-dihydro-1H-1,2,4-triazol-5-one 4a
1
6
tuted benzenoid ring); H NMR (200 MHz, DMSO-d ) d 1.21 (t, 3H,
4
CH , J ¼ 7.81 Hz), 2.69 (q, 2H, CH , J ¼ 7.42 Hz), 3.81 (s, 3H,
3
2
Yield 3.24 g (88.52%). m.p. 1408C. IR (KBr) n 3178 (NH), 1766, 1709
OCH
3
), 3.99 (s, 2H, COCH
2
), 7.21–7.64 (m, 8H, Ar-H), 9.70
ꢁ
1
13
(
tuted benzenoid ring); H NMR (200 MHz, DMSO-d
C–O), 1600 (C–N), 1269 (COO), 762 and 698 cm (monosubsti-
(s, 1H, N¼CH), 11.90 (s, 1H, NH); C NMR (50 MHz, DMSO-d
6
)
1
6
) d 2.27 (s, 3H,
CH ), 3.80 (s, 3H, OCH ), 3.97 (s, 2H, COCH ), 7.19–7.58 (m, 8H,
d 9.97 (CH
3 2 2 3
), 18.49 (CH ), 55.82 (COCH ), 55.90 (OCH ), 111.44,
3
3
2
120.37, 123.37, 127.07, 128.45 (2C), 129.49 (2C), 132.52, 133.75,
Table 3. Antibacterial and antifungal activity of selected compounds (MIC in mg/mL).
Yeast
Bacteria
Test materials
C. albicans
C. krusei
C. parapsilosis
S. aureus
E. coli
P. aeruginosa
E. faecalis
ATCC 90028
ATCC 6258
ATCC 90018
ATCC 29213
ATCC 25922
ATCC 27853
ATCC 29212
4
4
4
4
4
4
4
5
5
5
5
5
a
b
c
d
e
f
g
a
d
e
f
128
128
256
256
256
256
256
256
256
256
256
256
1
64
128
128
128
128
64
128
256
256
256
256
256
128
256
256
128
128
256
0.5
>1024
512
512
256
128
512
256
512
1024
512
512
512
>1024
512
512
256
256
256
128
256
256
128
256
512
256
128
256
128
128
128
32
128
64
512
512
512
256
512
512
128
64
64
256
128
256
256
256
128
128
128
128
16
>1024
512
>1024
g
Fluconazol
Ciprofloxacin
n
0.5
0.015
1
2
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
H. Y u¨ ksek et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
1
1
3
41.67, 151.17 (arom-C), 148.07 (triazole C
52.90 (triazole C
08 (14853), 262 (7764), 220 (15321) nm.
3
), 151.34 (N–CH),
5 2 5
), 169.33 (COO); UV (C H OH) lmax (log e)
3
-Phenyl-4-(3-methoxy-4-phenylacetoxybenzylidenamino)-
4,5-dihydro-1H-1,2,4-triazol-5-one 4g
Yield 3.87 g (90.65%). m.p. 1568C. IR (KBr) n 3196 (NH), 1769, 1717
ꢁ1
(
C–O), 1600, 1584 (C–N), 1273 (COO), 766 and 696 cm (mono-
1
substituted benzenoid ring); H NMR (200 MHz, DMSO-d
3
-n-Propyl-4-(3-methoxy-4-phenylacetoxybenzylidenamino)-
,5-dihydro-1H-1,2,4-triazol-5-one 4c
6
) d 3.80
(
(
s, 3H, OCH
s, 1H, N–CH), 12.45 (s, 1H, NH); C NMR (50 MHz, DMSO-d
3 2
), 3.98 (s, 2H, COCH ), 7.21–7.53 (m, 11H, Ar-H), 9.66
3
4
1
6
)
Yield 3.09 g (78.53%). m.p. 1408C. IR (KBr) n 3179 (NH); 1760, 1701
ꢁ1
2 3
d 48.20 (COCH ), 55.72 (OCH ), 111.14, 120.82, 123.39, 126.47,
127.00, 127.95 (2C), 128.39 (2C), 128.43 (2C), 129.42 (2C), 130.10,
(
C–O), 1615, 1595 (C–N), 1273 (COO), 758 and 703 cm (mono-
1
substituted benzenoid ring); H NMR (200 MHz, DMSO-d
6
) d 0.94
, J ¼ 7.52 Hz), 3.80
, J ¼ 7.25 Hz), 3.98 (s, 2H, COCH ),
.22 (d, 1H, Ar-H, J ¼ 8.32 Hz), 7.28–7.46 (m, 6H, Ar-H), 7.57
d, 1H, Ar-H, J ¼ 8.86 Hz), 9.70 (s, 1H, N–CH), 11,88 (s, 1H,
NH); UV (C OH) lmax (log e) 308 (10905), 264 (7878), 216
14540) nm; Anal. calcd. for C21 (394.43): C, 63.94;
H, 5.62; N, 14.20. Found: C, 63.13; H, 5.46; N, 13.69.
132.31, 133.69, 141.83, 151.14 (arom-C), 144.51 (triazole C
51.26 (N–CH), 155.17 (triazole C
1 ), 169.27 (COO); UV (C OH)
lmax (log e) 310 (5497), 266 (7524), 212 (15020) nm.
3
),
(
t, 3H, CH
3
, J ¼ 7.52 Hz), 1.67 (sext., 2H, CH
2
5
2 5
H
(
s, 3H, OCH
3
), 2.64 (q, 2H, CH
2
2
7
(
2 5
H
General procedure for the synthesis of compounds 5
(
22 4 4
H N O
The corresponding compound 4 (0.01 mol) was refluxed with
acetic anhydride (20 mL) for 0.5 h. After the addition of absolute
ethanol (100 mL), the mixture was refluxed for 1 h. Evaporation
of the resulting solution at 40–458C in vacuo and several recrys-
tallizations of the residue from EtOH gave pure compounds 5 as
colorless needles.
3
-Benzyl-4-(3-methoxy-4-phenylacetoxybenzylidenamino)-
4
,5-dihydro-1H-1,2,4-triazol-5-one 4d
Yield 3.71 g (83.95%). m.p. 1608C. IR (KBr) n 3174 (NH), 1747, 1705
ꢁ1
(
C–O), 1594 (C–N), 1275 (COO), 758 and 703 cm monosubsti-
1
1-Acetyl-3-methyl-4-(3-methoxy-4-phenylacetoxybenzyl-
tuted benzenoid ring); H NMR (200 MHz, DMSO-d
OCH ), 3.81 (s, 2H, CH ), 3.96 (s, 2H, COCH ), 7.17–7.58 (m, 13H,
Ar-H), 9.65 (s, 1H, N–CH), 12,02 (s, 1H, NH); UV (C
OH) lmax
log e) 310 (11577), 264 (7970), 218 (16642) nm; Anal. calcd.
for C25 (442.47): C, 67.86; H, 5.01; N, 12.66. Found: C,
6.96; H, 5.03; N, 12.39.
6
) d 3.79 (s, 3H,
3
2
2
idenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one 5a
Yield 3.82 g (93.86%). m.p. 1698C. IR (KBr) n 1781, 1765 (C–O),
2
H
5
ꢁ1
(
1600, 1585 (C–N), 1301 (COO), 748 and 708 cm (monosubsti-
1
tuted benzenoid ring); H NMR (200 MHz, DMSO-d ) d 2.35 (s, 3H,
H
22
N
4
O
4
6
6
CH ), 2.48 (s, 3H, COCH ), 3.82 (s, 3H, OCH ), 3.99 (s, 2H, COCH ),
3
3
3
2
1
3
7
.27–7.40 (m, 7H, Ar-H), 9.57 (s, 1H, N–CH); C NMR (50 MHz,
DMSO-d ) d 11.15 (CH ), 23.39 (COCH ), 48.20 (COCH ), 55.90
), 111.45, 120.95, 123.38, 127.00, 128.39 (2C), 129.42
2C), 131.89, 133.68, 142.04, 147.78 (arom-C), 146.68
triazole ), 166.01
), 151.19 (N–CH), 154.89 (triazole
COCH ), 169.23 (COO); UV (C OH) lmax (log e) 308 (11857),
98 (12414), 258 (10241), 218 (16741) nm.
6
3
3
2
3-p-Methylbenzyl-4-(3-methoxy-4-phenylacetoxybenzyl-
idenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one 4e
(
(
(
(
OCH
3
Yield 4.19 g (91.89%). m.p. 1648C. IR (KBr) n 3166 (NH), 1756, 1707
C
3
C
5
ꢁ1
(
zenoid ring), 762 and 690 cm (monosubstituted benzenoid
C–O), 1598 (C–N), 1272 (COO), 830 cm (1,4-disubstituted ben-
3
2 5
H
ꢁ
1
2
1
ring); H NMR (200 MHz, DMSO-d
6
) d 2.23 (s, 3H, CH
), 3.98 (s, 2H, COCH ), 7.08–7.49 (m,
2H, Ar-H), 9.65 (s, 1H, N–CH), 12.03 (s, 1H, NH); C NMR
) d 20.52 (CH ), 30.76 (CH Ph), 48.10 (COCH ),
), 110.47, 121.03, 123.28, 126.99, 128.39 (2C), 128.42
3
), 3.81 (s,
3
1
H, OCH
3
), 4.01 (s, 2H, CH
2
2
1
3
1-Acetyl-3-benzyl-4-(3-methoxy-4-phenylacetoxybenzyl-
(
50 MHz, DMSO-d
6
3
2
2
idenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one 5d
Yield 4.26 g (88.06%). m.p. 1208C. IR (KBr) n 1769, 1730, 1698
55.80 (OCH
3
ꢁ1
(
2C), 128.52 (2C), 128.98 (2C), 132.43, 132.67, 133.69, 135.74,
(C–O), 1600, 1580 (C–N), 1300 (COO), 754 and 690 cm (mono-
1
substituted benzenoid ring); H NMR (200 MHz, DMSO-d ) d 2.51
1
1
3
41.64, 150.80 (arom-C), 146.34 (triazole C
52.13 (triazole C ), 169.29 (COO); UV (C
10 (9286), 268 (5785), 210 (16603) nm. Anal. calcd.
(456.50): C, 68.40; H, 5.29; N, 12.27. Found: C,
6.87; H, 5.26; N, 12.14.
3
), 151.13 (N–CH),
6
5
2
H
5
OH) lmax (log e)
(s, 3H, COCH ), 3.81 (s, 3H, OCH ), 4.15 (s, 2H, CH ), 3.99 (s, 2H,
3
2
COCH ), 7.24–7.38 (m, 11H, Ar-H), 7.51 (s, 1H, Ar-H), 9.54 (s, 1H,
N–CH); C NMR (50 MHz, DMSO-d ) d 23.48 (COCH ), 30.61
6 3
2 2 3
(CH Ph), 48.60 (COCH ), 55.85 (OCH ), 110.60, 121.49, 123.34,
3
2
1
3
for C26
6
H
24
N
4
O
4
1
26.91, 126.99, 128.38 (2C), 128.46 (2C), 128.84 (2C), 129.42
2C), 131.90, 133.66, 134.67, 142.05, 148.19 (arom-C), 147.94
triazole ), 165.96
), 151.14 (N–CH), 154.01 (triazole
COCH ), 169.24 (COO); UV (C OH) lmax (log e) 310 (10212),
98 (10394), 258 (7960), 216 (17870) nm.
(
(
(
3-p-Chlorobenzyl-4-(3-methoxy-4-phenylacetoxybenzyl-
idenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one 4f
C
3
C
5
3
2 5
H
Yield 4.03 g (84.58%). m.p. 1658C. IR (KBr) n 3166 (NH), 1756, 1708
2
(
zenoid ring), 749 and 690 cm (monosubstituted benzenoid
C–O), 1600, 1585 (C–N), 1273 (COO), 828 (1,4-disubstituted ben-
ꢁ
1
1
1-Acetyl-3-p-methylbenzyl-4-(3-methoxy-4-
phenylacetoxybenzylidenamino)-4,5-dihydro-1H-1,2,4-
ring); H NMR (200 MHz, DMSO-d
s, 2H, CH ), 3.98 (s, 2H, COCH ), 7.23–7.46 (m, 12H, Ar-H),
.66 (s, 1H, N–CH), 12.07 (s, 1H, NH), C NMR (50 MHz,
DMSO-d ) d 30.49 (CH Ph), 48.15 (COCH ), 55.84 (OCH ), 110.59,
21.01, 123.30, 124.70, 127.00, 128.39 (3C), 129.43 (2C), 130.60
2C), 131.36, 132.37, 133.71, 134.79, 141.70, 151.13 (arom-C),
45.86 (triazole C ), 169.29
), 151.13 (N–CH), 152.35 (triazole C
COO); UV (C OH) lmax (log e) 310 (5818), 270 (2760), 216
13935) nm.
6 3
) d 3.80 (s, 3H, OCH ), 4.07
(
9
2
2
1
3
triazol-5-one 5e
Yield 4.39 g (88.31%). m.p. 1128C. IR (KBr) n 1769, 1732 (C–O),
6
2
2
3
1
(
1604, 1582 (C–N), 1309 (COO), 830 (1,4-disubstituted benzenoid
ꢁ1
ring), 755 and 695 cm
1
(monosubstituted benzenoid ring);
H NMR (200 MHz, DMSO-d ) d 2.24 (s, 3H, CH ), 2.51 (s, 3H,
1
3
5
6
3
(
(
2
H
5
3 3 2
COCH ), 3.82 (s, 3H, OCH ), 4.10 (s, 2H, CH ), 3.99 (s, 2H,
2
COCH ), 7.14–7.40 (m, 11H, Ar-H), 7.51 (s, 1H, Ar-H), 9.54 (s,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
4,5-Dihydro-1H-1,2,4-triazol-5-one Derivatives
9
H, N–CH); 13C NMR (50 MHz, DMSO-d
COCH ), 30.64 (CH Ph), 48.21 (COCH
1
(
6
) d 20.55 (PhCH
3
), 23.51
), 55.87 (OCH ), 110.63,
trophometer. Higher absorbance of the reaction mixture indi-
cated greater reducing power.
3
2
2
3
1
1
21.57, 123.38, 127.01, 128.41 (2C), 128.73 (2C), 129.05 (2C),
29.43 (2C), 131.55, 131.95, 133.69, 136.06, 147.07, 148.36
Free radical scavenging activity
Free radical scavenging activity of compounds was measured via
(arom-C), 147.97 (triazole
(triazole C ), 166.01 (COCH ), 169.26 (COO); UV (C
(log e) 310 (18080), 298 (14340), 260 (11446), 220 (24377) nm.
C
3
), 151.17 (N–CH), 152.10
5
3
2 5
H
OH) lmax
ꢀ
DPPH by using the method of Blois [44]. Briefly, 0.1 mM solution
of DPPH in ethanol was prepared, and this solution (1 mL) was
ꢀ
added to sample solutions in DMSO (3 mL) at different concen-
trations (50–250 mg/mL). The mixture was shaken vigorously and
allowed to remain at the room temperature for 30 min. Then,
the absorbance was measured at 517 nm in a spectrophometer.
The lower absorbance of the reaction mixture indicated higher
1
-Acetyl-3-p-chlorobenzyl-4-(3-methoxy-4-phenylacetoxy-
benzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one 5f
Yield 4.35 g (84.06%). m.p. 1128C. IR (KBr) n 1773, 1730, 1699
(
C–O), 1609, 1582 (C–N), 1308 (COO), 804 (1,4-disubstituted ben-
zenoid ring), 744 and 685 cm (monosubstituted benzenoid
ꢁ
1
ꢀ
free radical scavenging activity. The DPPH concentration (mM)
in the reaction medium was calculated from the following cali-
bration curve and determined by linear regression (R: 0.997):
1
ring); H NMR (200 MHz, DMSO-d
6
) d 2.51 (s, 3H, COCH
), 3.99 (s, 2H, COCH ), 7.25–7.50
m, 12H, Ar-H), 9.55 (s, 1H, N–CH); C NMR (50 MHz, DMSO-d
), 30.61 (CH Ph), 51.62 (COCH ), 55.85 (OCH
10.67, 121.48, 123.35, 126.97, 128.38 (4C), 129.41 (2C), 130.76
2C), 131.60, 131.86, 133.69, 136.40, 142.08, 148.70 (arom-C),
47.88 (triazole C ), 165.90
), 151.16 (N–CH), 154.19 (triazole C
COCH ), 169.23 (COO); UV (C OH) lmax (log e) 310 (11660), 298
12160), 258 (9960), 226 (20604), 218 (19950) nm. Anal. calcd.
Cl (519.96): C, 62.36; H, 4.65; N, 10.77. Found: C,
1.47; H, 4.64; N, 10.90.
3
), 3.81
(
(
s, 3H, OCH
3
), 4.17 (s, 2H, CH
2
2
1
3
ꢀ
6
)
Absorbance ¼ (0.0003 ꢃ DPPH ) ꢁ 0.0174
d 23.47 (COCH
1
(
1
(
(
3
2
2
3
),
The capability to scavenge the DPPH radical was calculated by
using the following equation: DPPH scavenging effect
ꢀ
(%) ¼ (A
trol reaction, and A
samples or standards.
0
ꢁ A
1
/A
0
) ꢃ 100, where A
0
is the absorbance of the con-
is the absorbance in the presence of the
3
5
1
3
2 5
H
24 4 5
for C27H N O
6
Metal chelating activity
The chelation of ferrous ions by the synthesized compounds and
standards was estimated by the method of Dinis et al. [45].
Shortly, the synthesized compounds (50–250 mg/mL) were added
2
to a 2 mM solution of FeCl (0.05 mL). The reaction was initiated
by the addition of 5 mM ferrozine (0.2 mL), and then the mixture
1-Acetyl-3-phenyl-4-(3-methoxy-4-phenylacetoxybenzyl-
idenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one 5g
Yield 4.03 g (85.78%). m.p. 1098C. IR (KBr) n 1772, 1733 (C–O),
ꢁ1
1
603, 1591 (C–N), 1305 (COO), 772 and 693 cm (monosubsti-
was shaken vigorously and left remaining at the room tempera-
ture for 10 min. After the mixture had reached equilibrium,
the absorbance of the solution was measured at 562 nm in a
spectrophotometer. All tests and analyses were carried out
in triplicate and averaged. The percentage of inhibition of
1
tuted benzenoid ring); H NMR (200 MHz, DMSO-d
COCH ), 3.80 (s, 3H, OCH ), 3.99 (s, 2H, COCH
m, 11H, Ar-H), 7.56–7.60 (m, 2H, Ar-H), 9.52 (s, 1H, N–CH);
6
) d 2.50 (s, 3H,
3
3
2
), 7.27–7.43
(
1
3
C NMR (50 MHz, DMSO-d
5.75 (OCH ), 111.27, 121.26, 123.52, 125.13, 127.00, 128.39
2C), 128.60 (2C), 128.65 (2C), 129.42 (2C), 131.30, 131.77,
33.65, 142.18, 148.08 (arom-C), 145.95 (triazole C ), 151.19
N–CH), 157.24 (triazole C
), 166.23 (COCH ), 169.26 (COO); UV
OH) lmax (log e) 310 (13030), 264 (18255), 234 (19990), 218
17451) nm; Anal. calcd. for C26 (470.48): C, 66.37; H,
.71; N, 11.90. Found: C, 65.24; H, 4.72; N, 12.17.
6 3 2
) d 23.54 (COCH ), 55.75 (COCH ),
2
þ
5
(
1
3
ferrozine–Fe complex formation was given by the formula:
Inhibition% ¼ (A ꢁ A /A ) ꢃ 100, where A is the absorbance of
the control, and A is the absorbance in the presence of the
0
1
0
0
3
1
(
(
(
5
3
samples or standards. The control did not contain compound
or standard.
2 5
C H
22 4 5
H N O
4
Potentiometric titrations
A Jenco model ion analyzer (Jenco, USA) and an Ingold pH elec-
trode (Mettler Toledo, Spain) were used for potentiometric
titrations. For each titrated compound, the 0.001 M solution
was separately prepared in each non-aqueous solvent. 0.05 M
TBAH solution in isopropyl alcohol, which is widely preferred
in the titration of acids, was used as titrant. The mV values were
obtained in pH-meter. Finally, the half-neutralization potentials
(HNP) values were determined by drawing the mL (TBAH)-mV
graphic.
Antioxidant activity
Chemicals
Butylated hydroxytoluene (BHT) was purchased from E. Merck.
Ferrous chloride, a-tocopherol, 1,1-diphenyl-2-picryl-hydrazyl
ꢀ
(
DPPH ), 3-(2-pyridyl)-5,6-bis(phenylsulfonic acid)-1,2,4-triazine
ferrozine), butylated hydroxyanisole (BHA), and trichloroacetic
acid (TCA) were bought from Sigma (Sigma–Aldrich GmbH).
(
Reducing power
0
The reducing power of the synthesized compounds was deter-
mined according to the method of Oyaizu [43]. Different con-
centrations of the samples (50–250 mg/mL) in DMSO (1 mL) were
mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potas-
sium ferricyanide (2.5 mL, 1%). The mixture was incubated at
Measurement of log k
The LC system consisted of a Spectra-SYSTEM P2000 gradient
pump, a Spectra SYSTEM SCM 1000 degasser, a Rheodyne manual
injector with a 20 mL injection loop and a Spectra SYSTEM
UV2000 detector (Thermo Separation Products, USA). The detec-
˚
5
08C for 20 min and afterwards a portion (2.5 mL) of trichloro-
tor was set at 254 nm. A Phenomenex Luna 5 mm C18 100 A LC
0
acetic acid (10%) was added to the mixture, which was centri-
fuged for 10 min at 1000g. The upper layer of solution (2.5 mL)
was mixed with distilled water (2.5 mL) and FeCl (0.5 mL, 0.1%),
3
column (250 ꢃ 4.6 mm) was used for elution. The log k values
were determined for each compound. The mobile phases were
made by mixing methanol with water in the proportions 100:0,
90:10, 85:15, 80:20, and 75:25 (v/v). The flow rate was 1.0 mL/min.
and then the absorbance at 700 nm was measured in a spec-
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

1
0
H. Y u¨ ksek et al.
Arch. Pharm. Chem. Life Sci. 2013, 000, 1–11
All measurements were made in, at least, duplicate. The average
reproducibility of each determination was better than 1.0%
[9] R. Kaliszan, P. Haber, T. Baczek, D. Siluk, Pure Appl. Chem.
2001, 73, 1465–1475.
0
relative. The capacity factors (k ) were determined using
0
[
[
[
[
[
10] H. Yuksek, A. Demirbas, A. Ikizler, C. B. Johansson, C. Celik,
A. A. Ikizler, Arzneim.-Forsch. 1997, 47, 405–409.
k ¼ (t
R
ꢁ t
0 0 R
)/t , where t is the retention time of the compound,
and t
0
is the void volume or the dead time. An aqueous solution
11] B. Kahveci, M. Ozil, E. Mentese, O. Bekircan, K. Buruk, Russ. J.
Org. Chem. 2008, 44, 1816–1820.
of uracil was used for the measurement of void volume. All the
chemical components were dissolved in methanol and diluted
with mobile phase before injection into the HPLC system so the
final concentration is 4 mg/mL.
12] N. Chidananda, B. Poojary, V. Sumangala, N. S. Kumari,
P. Shetty, T. Arulmoli, Eur. J. Med. Chem. 2012, 51, 124–136.
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E. De Clercq, X. Y. Liu, Lett. Drug Des. Discov. 2013, 10, 27–34.
Microbiology: Antibacterial screening
The antibacterial activity of the compounds 4a–g, 5a and 5d–g
were tested against four bacteria including two Gram positive
14] M. A. Henen, S. A. A. El Bialy, F. E. Goda, M. N. A. Nasr, H. M.
Eisa, Med. Chem. Res. 2012, 21, 2368–2378.
(
Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212)
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Med. Chem. 2009, 44, 1057–1066.
and two Gram negative microorganisms (Escherichia coli
ATCC25922, Pseudomonas aeruginosa ATCC 27853) and for antifun-
gal activities against three yeasts (Candida albicans ATCC 90028,
Candida krusei ATCC 6258, Candida parapsilosis ATCC 90018) by
using ciprofloxacin and fluconazole as reference compounds.
Antimicrobial activities were determined as minimum inhibi-
tory concentration (MIC) values which were determined by broth
microdilution method reported by the Clinical and Laboratory
Standards Institute (CLSI) [46, 47]. Antibacterial activity test was
performed in Mueller Hinton Broth (Difco, USA). RPMI-1640
medium with L-glutamine (ICN-Flow, USA) was used as the
culture medium for antifungal activity test in 378C. The
[16] A. Uzgoren-Baran, B. C. Tel, D. Sarigol, E. I. Ozturk, I.
Kazkayasi, G. Okay, M. Ertan, B. Tozkoporan, Eur. J. Med.
Chem. 2012, 57, 398–406.
[
17] M. Alkan, H. Yuksek, O. Gursoy-Kol, M. Calapoglu, Molecules
2
008, 13, 107–121.
[
18] O. G. Kol, H. Yuksek, Eur. J. Chem. 2010, 7, 123–136.
19] H. Yuksek, O. G. Kol, Turk. J. Chem. 2008, 32, 773–784.
[
[20] H. Yuksek, O. Gursoy-Kol, G. Kemer, Z. Ocak, B. Anil, Indian J.
Heterocycl. Chem. 2011, 20, 325–330.
[21] S. Bahceci, H. Yuksek, Z. Ocak, I. Azakli, M. Alkan, M. Ozdemir,
Collect. Czech. Chem. Commun. 2002, 67, 1215–1222.
5
inoculum densities were approximately 5 ꢃ 10 cfu/mL and
3
0
.5–2.5 ꢃ 10 cfu/mL for bacteria and fungi, respectively. The
[22] S. Bahceci, H. Yuksek, Z. Ocak, C. Koksal, M. Ozdemir, Acta
Chim. Slov. 2002, 49, 783–794.
MIC values were recorded as the lowest concentrations of the
substances that had no visible turbidity. In microbial studies, all
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[
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