Synthesis, in vitro antioxidant activity, and physicochemical properties of novel 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives

Article abstract of DOI:10.1016/j.molliq.2015.02.038

In this study, eight new 3-alkyl(aryl)-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-1H-1,2,4-triazol-5-one (4) compounds were synthesized by the reactions of 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (3) with 4-(4-methylbenzoxy)benzaldehyde (1). The eight compounds were characterized using IR, ¹H NMR, ¹³C NMR, and UV spectral data. In addition, the synthesized compounds were analyzed for their potential in vitro antioxidant activities, including reducing power, free radical scavenging and metal chelating activity. These antioxidant activities were compared to those from standard antioxidants, such as EDTA, BHA, BHT and α-tocopherol. Moreover, compounds 4 were titrated potentiometrically with tetrabutylammonium hydroxide in four non-aqueous solvents (isopropyl alcohol, tert-butyl alcohol, acetone and N,N-dimethylformamide). Thus, the half-neutralization potential values and the corresponding pK_a values were determined in all cases. In addition, the lipophilicity of the synthesized compounds was investigated using HPLC. Kinetic parameters of the complexes were obtained for each stage of thermal degradation using the Coats-Redfern and Horowitz-Metzger methods.

Full text of DOI:10.1016/j.molliq.2015.02.038

Journal of Molecular Liquids 206 (2015) 359–366
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Synthesis, in vitro antioxidant activity, and physicochemical properties of
novel 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives
b
c
Haydar Yüksek a, Ebru Koca a, Özlem Gürsoy-Kol a, , Onur Akyıldırım , Mustafa Çelebier
⁎
a
Department of Chemistry, Faculty of Science and Letters, Kafkas University, 36100 Kars, Turkey
Department of Chemical Engineering, Faculty of Engineering and Architecture, Kafkas University, 36100 Kars, Turkey
Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara, Turkey
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 October 2014
Received in revised form 6 February 2015
Accepted 25 February 2015
Available online 4 March 2015
In this study, eight new 3-alkyl(aryl)-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-1H-1,2,4-triazol-
5-one (4) compounds were synthesized by the reactions of 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-
triazol-5-ones (3) with 4-(4-methylbenzoxy)benzaldehyde (1). The eight compounds were characterized
using IR, 1H NMR, 13C NMR, and UV spectral data. In addition, the synthesized compounds were analyzed for
their potential in vitro antioxidant activities, including reducing power, free radical scavenging and metal
chelating activity. These antioxidant activities were compared to those from standard antioxidants, such
as EDTA, BHA, BHT and α-tocopherol. Moreover, compounds 4 were titrated potentiometrically with
tetrabutylammonium hydroxide in four non-aqueous solvents (isopropyl alcohol, tert-butyl alcohol, ace-
tone and N,N-dimethylformamide). Thus, the half-neutralization potential values and the corresponding pKa
values were determined in all cases. In addition, the lipophilicity of the synthesized compounds was investigated
using HPLC. Kinetic parameters of the complexes were obtained for each stage of thermal degradation using the
Coats–Redfern and Horowitz–Metzger methods.
Keywords:
1,2,4-Triazol-5-one
Acylation
Antioxidant activity
Schiff base
Syntheses
Physicochemical properties
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
activity on many cancer types, such as leukemia, non-small cell lung,
colon, melanoma, ovarian, renal, prostate, and breast cancers. Several
Antioxidants have the capacity to protect organisms and cells from
damage induced by oxidative stress; therefore, considerable research
has been conducted to examine this feature. Novel compounds have
attracted scientists' interest in recent years. Natural sources that provide
the active components for preventing or reducing the impact of oxida-
tive stress on cells have been used [1]. Exogenous chemicals and endog-
enous metabolic processes in the human body or in food systems might
produce highly reactive free radicals, especially oxygen, which are capa-
ble of oxidizing biomolecules that can result in cell death and tissue
damage. Oxidative damage has a pathological role in serious human
diseases (e.g., emphysema, cirrhosis, atherosclerosis and arthritis).
Furthermore, a variety of pathophysiological processes, such as inflam-
mation, diabetes, genotoxicity and cancer, stem from the excessive gen-
eration of reactive oxygen species (ROS) induced by various stimuli that
exceed the antioxidant capacity of the organism [2].
in vitro studies were performed for this purpose in recent years [3–7].
The anticancer activity of 1,2,4-triazole derivatives might be associated
with their antioxidant activity. Additionally, 1,2,4-triazole and 4,5-
dihydro-1H-1,2,4-triazol-5-one derivatives have a broad spectrum of
biological activities [8–18]. In addition, several studies involving the
synthesis of N-arylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-one
derivatives are available in the literature [15–21].
In the present study, the following antioxidant activities were
explored using the newly synthesized compounds: 1,1-diphenyl-2-
picryl-hydrazyl (DPPH.) free radical scavenging, reducing power and
metal chelating activities. Furthermore, several 1,2,4-triazole and 4,5-
dihydro-1H-1,2,4-triazol-5-one derivatives were titrated potentiomet-
rically with tetrabutylammonium hydroxide (TBAH) in non-aqueous
solvents to determine their pKa values [15–23]. The hydrophobicity,
meaning the solubility of synthesized compounds in aqueous solutions,
was also investigated. The concentration of a non-ionized compound in
two different solutions is the partition coefficient. The partition coeffi-
cient (log P) of any ionizable solute could be measured in the aqueous
phase in which the pH value was adjusted for its non-ionized form.
The ratio of the concentrations of non-ionized solute in the solvents is
calculated according to the log P values. The log P value is a measure
of lipophilicity and is not pH-dependent. In the HPLC analysis, the
Even several natural sources of these active components have been
used to synthesize and obtain effective new antioxidative compounds.
It is well-known that 1,2,4-triazole derivatives present an antitumor
⁎
Corresponding author.
E-mail address: ozlemgursoy@gmail.com (Ö. Gürsoy-Kol).
http://dx.doi.org/10.1016/j.molliq.2015.02.038
0167-7322/© 2015 Elsevier B.V. All rights reserved.

360
H. Yüksek et al. / Journal of Molecular Liquids 206 (2015) 359–366
capacity factor (k′), which is a measure of the migration rate of an ana-
lyte on a column, was extrapolated from the binary eluents to 100%
water to find the log k′w values. It is reported that log k′w values could
2.2.2. 3-Ethyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-1H-
1,2,4-triazol-5-one (4b)
Yield: 3.40 g (97%); mp: 180–182 °C; IR (KBr, υ, cm−1): 3160 (NH),
1739, 1700 (C_O), 1595 (C_N), 1264 (COO), 832 (1,4-disubstituted
benzenoid ring); 1H NMR (400 MHz, DMSO-d6): δ 1.23 (t, 3H,
CH2CH3), 2.44 (s, 3H, PhCH3), 2.70 (q, 2H, CH2CH3), 7.43–7.46 (m, 4H,
Ar-H), 7.93–7.96 (m, 2H, Ar-H), 8.04–8.07 (m, 2H, Ar-H), 9.77 (s, 1H,
N_CH), 11.88 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): δ 10.57
(CH2CH3), 19.02 (CH2CH3), 21.76 (PhCH3), 123.20 (2C), 126.37 (arom-
C), 129.45 (2C), 130.06 (2C), 130.41 (2C), 131.84, 145.28, 153.21
(arom-C), 148.54 (triazole C3), 151.84 (N_CH), 153.38 (triazole C5),
164.79 (COO); UV λmax (ε): 292 (13.503), 252 (15.579) nm.
successfully replace the n-octanol–water partition coefficient (log Po/w
)
[24].
Because pKa and lipophilicity are highly related to the solubility of
these compounds in different liquid systems and the ability or inability
for them to cross the cell membrane, these parameters are responsible
for the biological activity in the body system [25,26]. Therefore, it is of
great importance to know the pKa and lipophilicity of any proposed
and potentially active compound to classify and understand its behavior
with respect to the permeability in body tissues and to understand the
compound's solubility in various liquid systems.
2.2.3. 3-n-Propyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-
1H-1,2,4-triazol-5-one (4c)
2. Experimental
Yield: 3.50 g (96%); mp: 171–173 °C; IR (KBr, υ, cm−1): 3207 (NH),
1731, 1689 (C_O), 1593 (C_N), 1259 (COO), 835 (1,4-disubstituted
benzenoid ring); 1H NMR (400 MHz, DMSO-d6): δ 1.02 (t, 3H,
CH2CH2CH3), 1.76 (sext, 2H, CH2CH2CH3), 2.50 (s, 3H, PhCH3), 2.72 (t,
2H, CH2CH2CH3), 7.48–7.51 (m, 4H, Ar-H), 7.99 (d, 2H, Ar-H), 8.11 (d,
2H, Ar-H), 9.82 (s, 1H, N_CH), 11.94 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): δ 13.96 (CH2CH2CH3), 19.40 (CH2CH2CH3), 21.75 (PhCH3),
27.19 (CH2CH2CH3), 123.20 (2C), 126.36 (arom-C), 129.43 (2C),
130.05 (2C), 130.41 (2C), 131.83, 145.22, 153.23 (arom-C), 147.39
(triazole C3), 151.78 (N_CH), 153.38 (triazole C5), 164.78 (COO); UV
2.1. Chemistry
Chemical reagents and all solvents used in this study were pur-
chased from Merck AG (Darmstadt, Germany), Sigma (Sigma-Aldrich
GmbH, Sternheim, Germany) and Fluka (Buchs, Switzerland). The
starting compounds 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-
triazol-5-ones 3 were prepared from the reactions of the corre-
sponding ester ethoxycarbonylhydrazones 2 with an aqueous solu-
tion of hydrazine hydrate as described in the literature [27,28].
Melting points were determined in open glass capillaries using a
WRS-2A Microprocessor melting-point apparatus (Liaoning, main-
land China) and are uncorrected. The IR spectra were obtained on
an ALPHA-P BRUKER FT-IR (Germany) spectrometer. 1H and 13C NMR
spectra were recorded in deuterated dimethyl sulfoxide with TMS
as an internal standard using a Bruker (Germany) spectrometer at
400 MHz and 100 MHz, respectively. UV absorption spectra were mea-
sured in 10 mm quartz cells between 200 and 400 nm using a PG Instru-
ments Ltd T80 UV/VIS (Leicestershire, United Kingdom) spectrometer.
λmax (ε): 294 (15.098), 256 (18.553) nm.
2.2.4. 3-Benzyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-
1H-1,2,4-triazol-5-one (4d)
Yield: 4.04 g (98%); mp: 197–198 °C; IR (KBr, υ, cm−1): 3155 (NH),
1738, 1693 (C_O), 1594 (C_N), 1265 (COO), 815 (1,4-disubstituted
benzenoid ring), 764 and 701 (monosubstituted benzenoid ring); 1H
NMR (400 MHz, DMSO-d6): δ 2.44 (s, 3H, PhCH3), 4.08 (s, 2H, CH2Ph),
7.22–7.34 (m, 5H, Ar-H), 7.42–7.45 (m, 4H, Ar-H), 7.91 (d, 2H, Ar-H),
8.05 (d, 2H, Ar-H), 9.73 (s, 1H, N_CH), 12.00 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6): δ 21.75 (PhCH3), 31.54 (CH2Ph), 123.16 (2C),
126.37 (arom-C), 127.21 (arom-C), 128.94 (2C), 129.28 (2C), 129.48
(2C), 130.05 (2C), 130.41 (2C), 131.77, 136.28, 145.22, 152.98 (arom-
C), 146.72 (triazole C3), 151.69 (N_CH), 153.39 (triazole C5), 164.78
(COO); UV λmax (ε): 292 (18.884), 256 (23.052) nm.
Extinction coefficients (ε) are expressed in L mol−1 cm−1
.
2.2. General procedure for the synthesis of compounds 4
4-Hydroxybenzaldehyde (0.01 mol) dissolved in ethyl acetate
(100 mL) was treated with 4-methylbenzoyl chloride (0.01 mol);
triethylamine (0.01 mol) was added slowly with stirring at 0–5 °C. Stir-
ring was continued for 1 h, followed by refluxing for 3 h and filtering.
The filtrate was evaporated in vacuo and the crude product was washed
with water and recrystallized from ethanol to afford compound 1. The
corresponding compound 3 (0.01 mol) was dissolved in acetic acid
(20 mL) and treated with 4-(4-methylbenzoxybenzaldehyde) 1
(0.01 mol). The mixture was refluxed for 2 h and subsequently
evaporated at 50–55 °C in vacuo. Several recrystallizations of the res-
idue from ethanol gave pure compounds 3-alkyl(aryl)-4-[4-(4-
methylbenzoxy)benzylidenamino]-4,5-dihydro-1H-1,2,4-triazol-5-
one 4 as colorless crystals.
2.2.5. 3-p-Methylbenzyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-
dihydro-1H-1,2,4-triazol-5-one (4e)
Yield: 4.08 g (96%); mp: 171–173 °C; IR (KBr, υ, cm−1): 3154 (NH),
1739, 1692 (C_O), 1595 (C_N), 1263 (COO), 820 (1,4-disubstituted
benzenoid ring); 1H NMR (400 MHz, DMSO-d6): δ 2.25 (s, 3H, PhCH3),
2.44 (s, 3H, PhCH3), 4.02 (s, 2H, CH2Ph), 7.12 (d, 2H, Ar-H), 7.22 (d,
2H, Ar-H), 7.42–7.45 (m, 4H, Ar-H), 7.91 (d, 2H, Ar-H), 8.05 (d, 2H, Ar-
H), 9.72 (s, 1H, N_CH), 11.98 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): δ 21.08 (CH2PhCH3), 21.76 (PhCH3), 31.15 (CH2Ph), 123.19
(2C), 126.38 (arom-C), 129.14 (2C), 129.50 (2C), 129.50 (2C), 130.06
(2C), 130.41 (2C), 131.79, 133.16, 136.27, 145.23, 152.98 (arom-C),
146.88 (triazole C3), 151.68 (N_CH), 153.38 (triazole C5), 164.80
(COO); UV λmax (ε): 292 (12.014), 256 (15.021) nm.
2.2.1. 3-Methyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-
1H-1,2,4-triazol-5-one (4a)
2.2.6. 3-p-Methoxybenzyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-
dihydro-1H-1,2,4-triazol-5-one (4f)
Yield: 3.26 g (97%); mp: 233 °C; IR (KBr, υ, cm−1): 3179 (NH), 1726,
1703 (C_O), 1599 (C_N), 1274 (COO), 833 (1,4-disubstituted benze-
noid ring); 1H NMR (400 MHz, DMSO-d6): δ 2.30 (s, 3H, CH3), 2.44 (s,
3H, PhCH3), 7.43–7.45 (m, 4H, Ar-H), 7.95 (d, 2H, Ar-H), 8.05 (d, 2H,
Ar-H), 9.77 (s, 1H, N_CH), 11.86 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): δ 11.61 (CH3), 21.76 (PhCH3), 123.17 (2C), 126.36 (arom-
C), 129.48 (2C), 130.06 (2C), 130.41 (2C), 131.80, 144.78, 153.15
(arom-C), 145.23 (triazole C3), 151.69 (N_CH), 153.37 (triazole C5),
164.78 (COO); UV λmax (ε): 290 (16.151), 254 (19.504), 220 (19.412) nm.
Yield: 4.36 g (99%); mp: 196–197 °C; IR (KBr, υ, cm−1): 3157 (NH),
1738, 1695 (C_O), 1594 (C_N), 1243 (COO), 828 (1,4-disubstituted
benzenoid ring); 1H NMR (400 MHz, DMSO-d6): δ 2.44 (s, 3H, PhCH3),
3.71 (s, 3H, PhOCH3), 4.00 (s, 3H, CH2Ph), 6.88 (d, 2H, Ar-H), 7.26 (d,
2H, Ar-H), 7.43–7.45 (m, 4H, Ar-H), 7.93 (d, 2H, Ar-H), 8.06 (d, 2H,
Ar-H), 9.73 (s, 1H, N_CH), 11.96 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): δ 21.76 (PhCH3), 30.68 (CH2Ph), 55.47 (OCH3), 114.34
(2C), 123.21 (2C), 126.37 (arom-C), 128.03, (arom-C), 129.51 (2C),
130.07 (2C), 130.35 (2C), 130.42 (2C), 131.80, 145.24, 153.00, 158.55

H. Yüksek et al. / Journal of Molecular Liquids 206 (2015) 359–366
361
(arom-C), 147.05 (triazole C3), 151.70 (N_CH), 153.39 (triazole C5),
164.81 (COO); UV λmax (ε): 292 (16.212), 254 (19.374) nm.
at 1000 ×g. The upper layer of the solution (2.5 mL) was mixed with dis-
tilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%) and then the absorbance
at 700 nm was measured in a spectrophotometer. A higher absorbance
of the reaction mixture indicated a greater reducing power.
2.2.7. 3-p-Chlorobenzyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-
dihydro-1H-1,2,4-triazol-5-one (4g)
Yield: 4.28 g (96%); mp: 210 °C; IR (KBr, υ, cm−1): 3169 (NH), 1729,
1705 (C_O), 1605 (C_N), 1259 (COO), 840 (1,4-disubstituted benze-
noid ring); 1H NMR (400 MHz, DMSO-d6): δ 2.44 (s, 3H, PhCH3), 4.09
(s, 2H, CH2Ph), 7.36–7.45 (m, 8H, Ar-H), 7.91 (d, 2H, Ar-H), 8.05 (d,
2H, Ar-H), 9.73 (s, 1H, N_CH), 12.03 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): δ 21.76 (PhCH3), 30.85 (CH2Ph), 123.20 (2C), 126.36
(arom-C), 128.87 (2C), 129.52 (2C), 130.06 (2C), 130.41 (2C), 131.24
(2C), 131.73 (arom-C), 131.90, 135.25, 145.24, 153.06 (arom-C),
146.41 (triazole C3), 151.67 (N_CH), 153.41 (triazole C5), 164.80
(COO); UV λmax (ε): 292 (17.171), 256 (21.127) nm.
2.3.2. Free radical scavenging activity
The free radical scavenging activity of the synthesized compounds
was determined. Briefly, a 0.1 mM solution of DPPH. in ethanol was
prepared, and this solution (1 mL) was added to sample solutions in
ethanol (3 mL) at various concentrations (50–250 μg/mL). The mixture
was shaken vigorously and allowed to remain at room temperature for
30 min. Next, the absorbance was measured at 517 nm in a spectropho-
tometer. The lower absorbance of the reaction mixture indicated a
higher free radical scavenging activity. The DPPH. concentration (mM)
in the reaction medium was calculated from the following calibration
curve and determined by linear regression (R2: 0.997): Absorbance =
(0.0003 × DPPH.) − 0.0174.
The capability to scavenge the DPPH radical was calculated
by using the following equation: DPPH. scavenging effect (%) =
(A0 − A1 / A0) × 100, where A0 is the absorbance of the control
reaction, and A1 is the absorbance in the presence of the samples or
standards.
2.2.8. 3-Phenyl-4-[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-
1H-1,2,4-triazol-5-one (4h)
Yield: 3.80 g (95%); mp: 216 °C; IR (KBr, υ, cm−1): 3140 (NH), 1738,
1692 (C_O), 1604 (C_N), 1260 (COO), 760 and 686 (monosubstituted
benzenoid ring); 1H NMR (400 MHz, DMSO-d6): δ 2.34 (s, 3H, PhCH3),
7.43–7.46 (m, 4H, Ar-H), 7.54–7.56 (m, 3H, Ar-H), 7.91–7.94 (m, 4H,
Ar-H), 8.05 (d, 2H, Ar-H), 9.70 (s, 1H, N_CH), 12.41 (s, 1H, NH); 13C
NMR (100 MHz, DMSO-d6): δ 21.76 (PhCH3), 123.30 (2C), 126.33
(arom-C), 127.10 (arom-C), 128.42 (2C), 129.03 (2C), 129.71 (2C),
130.05 (2C), 130.41 (2C), 130.60, 131.59, 145.07, 156.18 (arom-C),
145.24 (triazole C3), 151.82 (N_CH), 153.58 (triazole C5), 164.78
(COO); UV λmax (ε): 302 (12.935), 254 (24.627) nm.
2.3.3. Metal chelating activity
The chelation of ferrous ions by the synthesized compounds and
standards was estimated. In the study, all of the compounds and the
standard antioxidants were dissolved in ethanol. Briefly, the synthe-
sized compounds (30–90 μg/mL) were added to a 2 mM solution of
FeCl2·4H2O (0.05 mL). The reaction was initiated by the addition of
5 mM ferrozine (0.2 mL), and then the mixture was shaken vigorously
and left standing at room temperature 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 run in
triplicate and averaged. The percentage of inhibition of ferrozine–Fe2+
2.3. Antioxidant activity: chemicals
Butylated hydroxytoluene (BHT) was purchased from E. Merck
(Darmstadt, Germany). Ferrous chloride, α-tocopherol, 1,1-diphenyl-
2-picryl-hydrazyl (DPPH.), 3-(2-pyridyl)-5,6-bis(phenylsulfonic acid)-
1,2,4-triazine (ferrozine), butylated hydroxyanisole (BHA), ethylenedi-
aminetetraacetic acid (EDTA) and trichloroacetic acid (TCA) were
bought from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany).
complex formation was given by the formula: % inhibition = (A0
−
A1 / A0) × 100, where A0 is the absorbance of the control, and A1 is the
absorbance in the presence of the samples or standards. The control
did not contain compound or standard.
2.3.1. Reducing power
The reducing power of the synthesized compounds was determined.
Different concentrations of the samples (50–250 μg/mL) in ethanol
(1 mL) were mixed with phosphate buffer (2.5 mL, 0.2 M, pH = 6.6)
and potassium ferricyanide (2.5 mL, 1%). The mixture was incubated
at 50 °C for 20 min and afterwards a portion (2.5 mL) of trichloroacetic
acid (10%) was added to the mixture, which was centrifuged for 10 min
2.4. Potentiometric titrations
A Jenco model ion analyzer (Jenco, USA) and an Ingold pH electrode
(Mettler Toledo, Spain) were used for potentiometric titrations. For
each compound that would be titrated, the 0.001 M solution was sepa-
rately prepared in each non-aqueous solvent. The 0.05 M solution of
O
Et3N
-HCl
OHC
OH
CH3
COCl
OHC
OC
CH3
+
1
N
NH
N
NH
R
C
+H2NNH2
-2C2H5OH
1/AcOH
-H2O
O
NNHCO2C2H5
O
R
N
R
N
O
OC2H5
OC
CH3
NH2
N=CH
2
3
4
a) R= CH3, b) R= CH2CH3, c) CH2CH2CH3, d) CH2C6H5, e) R= CH2C6H4.CH3 (p-),
f) R= CH2C6H4.OCH3 (p-), g) R= CH2C6H4.Cl (p-), h) R= C6H5
Scheme 1. Synthetic route for compounds 1, 3 and 4.

362
H. Yüksek et al. / Journal of Molecular Liquids 206 (2015) 359–366
Fig. 1. Scavenging effect of compounds 4a–h, BHT, BHA and α-tocopherol at different con-
centrations (12.5–25–37.5 μg/mL).
TBAH in isopropyl alcohol, which is widely used in the titration of acids,
was used as titrant. The mV values that were obtained by the pH-
meter were recorded. Finally, the HNP values were determined by
plotting the mL (TBAH)-mV graphic.
Fig. 3. Potentiometric titration curves of 0.001 M solutions of compound 4b titrated with
0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, DMF and acetone at 25 °C.
3. Results and discussion
2.5. HPLC analysis for determination of log k′w values
3.1. Chemistry
The LC system consisted of a Spectra-SYSTEM P2000 gradient pump,
a Spectra SYSTEM SCM 1000 degasser, a Rheodyne manual injector with
a 20 μL injection loop and a Spectra SYSTEM UV2000 detector (Thermo
Separation Products, USA). The detector was set at 260 nm. A
Phenomenex Luna 5 mm C18 100 A° LC column (250 × 4.6 mm)
was used for elution. The logk′ values were determined for each
compound. The mobile phases were prepared by mixing methanol
with water in the proportions 100:0, 95:5, 90:10, 85:15, and 80:20
(v/v). The flow rate was 0.8 mL/min. All measurements were made
at least in duplicate. The average reproducibility of each determina-
tion was better than 1.0% relative. The capacity factors (k′) were
determined using k′ = (tR − t0) / t0, where tR is the retention time
of the compound, and t0 is the void volume or the dead time. An
aqueous solution of uracil was used for the measurement of void vol-
ume. All the chemical components were dissolved in methanol and
diluted with the mobile phase before injection into the HPLC system
4-(4-Methylbenzoxy)benzaldehyde 1, which was recently reported
[29], was obtained from the reaction of 4-hydroxybenzaldehyde with
4-methylbenzoyl chloride by using triethylamine. The 3-alkyl(aryl)-4-
[4-(4-methylbenzoxy)benzylidenamino]-4,5-dihydro-1H-1,2,4-triazol-
5-ones 4a–h were synthesized by the reactions of compounds 3-
alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones 3a–h with 4-
(4-methylbenzoxy)benzaldehyde 1 (Scheme 1).
The structures of eight new 3-alkyl(aryl)-4-[4-(4-methylbenzoxy)
benzylidenamino]-4,5-dihydro-1H-1,2,4-triazol-5-one 4a–h com-
pounds were identified by using IR, 1H NMR, 13C NMR and UV data.
3.2. Biological activity
3.2.1. Antioxidant activity
The antioxidant activities of eight new compounds 4a–h were iden-
tified. In the study, all of the compounds and the standard antioxidants
were dissolved in ethanol. If the compounds were not soluble in etha-
nol, DMSO was used for the compounds and the standards to compare
their activities in the same conditions. Several methods are used to
determine antioxidant activities. The methods used in the present
study are given below.
for a final concentration of 10 μg mL−1
.
Table 1
The HNP and the corresponding pKa values of compounds 4a–h in isopropyl alcohol,
tert-butyl alcohol, DMF and acetone.
Comp.
Isopropyl
alcohol
tert-Butyl
alcohol
DMF
Acetone
HNP
pKa
HNP
pKa
HNP
pKa
HNP
pKa
(mV)
(mV)
(mV)
(mV)
4a
4b
4c
4d
4e
4f
–
–
–
–
−477
−352
–
18.37
15.33
–
−380
15.83
12.18
–
–
–
–
–
−480
−
–
–
–
–
–
–
–
–
–
–
−99
–
8.91
–
–
–
–
–
−415
–
16.84
–
–
–
−364
–
13.49
–
−350
–
15.17
–
4g
4h
−308
–
14.32
–
−366
−269
15.72
13.33
Fig. 2. Metal chelating effect from different amounts of compounds 4a–h, EDTA and α-
tocopherol on ferrous ions.
–
–
–
–

H. Yüksek et al. / Journal of Molecular Liquids 206 (2015) 359–366
363
Table 2
used as a substrate to evaluate an antioxidative activity [36]. In this
study, antiradical activities of compounds and standard antioxidants
such as BHT, BHA and α-tocopherol were determined by using
the DPPH. method. Scavenging effect values of compounds 4 with
BHT, BHA and α-tocopherol at different concentrations are given in
Fig. 1. The newly synthesized compounds showed no radical scavenger
activity.
Measured log k′w values for the synthesized compounds.
Sa
b
Compound
kw
4a
4b
4c
4d
4e
4f
16.89499
25.35267
35.74553
41.41959
63.47511
38.31554
64.50168
39.05277
−0.17899
−0.27037
−0.38391
−0.44683
−0.68821
−0.41304
−0.70133
−0.42058
4g
4h
3.2.1.3. Ferrous ion chelating activity. The chelating effect towards ferrous
ions by the compounds and standards was determined according to the
method of Dinis [37]. Ferrozine can quantitatively form complexes
with Fe2+. In the presence of chelating agents, complex formation is
disrupted, resulting in the loss of the complexes' red color. Therefore,
color reduction measurements can serve as an estimate of the chelating
activity of the coexisting chelator [38]. Transition metals have a pivotal
role in the generation of free oxygen radicals in living organisms. Ferric
iron (Fe3+) is the relatively biologically inactive form of iron. However,
it can be reduced to the active Fe2+ form, depending on such conditions
as pH [39], and then oxidized again. Products of Fenton-type reactions
or Haber–Weiss reactions can interact with superoxide anions resulting
in hydroxyl radicals. The production of these highly active compounds
may lead to lipid peroxidation, protein modification and DNA damage.
Chelating agents may not activate the metal ions and in turn potentially
inhibit metal-dependent processes [40]. Additionally, the production of
highly active ROS, such as O2•−, H2O2 and OH•, is also catalyzed by free
iron though the Haber–Weiss reaction as shown below:
a
S is the slope.
b
log k′w is the intercept of the plot of log k′ versus φ (the volume percentages are
of methanol in the mobile phase).
3.2.1.1. Total reductive capability using the potassium ferricyanide
reduction method. The reductive capabilities of compounds are assessed
by the extent of conversion of the Fe3+/ferricyanide complex to the
Fe2+/ferrous form using the method of Oyaizu [30]. The reducing pow-
ers of the compounds were observed at different concentrations, and
results were compared with BHA, BHT and α-tocopherol. The reducing
capacity of a compound may serve as a significant indicator of its
potential antioxidant activity [31]. The antioxidant activity has been
attributed to various mechanisms, among which are the prevention of
chain initiation, binding of transition metal ion catalysts, decomposition
of peroxides, prevention of continued hydrogen abstraction, reductive
capacity and radical scavenging [32]. In this study, all of the compounds
had a lower absorbance than the standard antioxidants. Hence, no
activity was observed for reducing metal ion complexes to their lower
oxidation state or for any electron transfer reaction. Therefore, the com-
pounds did not exhibit a reductive activity.
O•2 + H2O2 → O2 + OH− + OH•.
Among the transition metals, iron is known as the most important
lipid oxidation pro-oxidant due to its high reactivity. The ferrous state
of iron accelerates lipid oxidation by breaking down the hydrogen and
lipid peroxides to form reactive free radicals via the Fenton reaction:
3.2.1.2. DPPH. radical scavenging activity. The free radical scavenging ac-
tivity of compounds was measured via DPPH. using the method of Blois
[33]. The stable DPPH radical scavenging model is a widely used method
to evaluate an antioxidant activity in a relatively short time compared
with other methods. The effect of antioxidants on DPPH radical scaveng-
ing was thought to be due to the antioxidant's hydrogen-donating
ability [34]. DPPH is a stable free radical and accepts an electron or
hydrogen radical to become a stable diamagnetic molecule [35]. The
reduction capability of DPPH radicals was determined by a decrease in
absorbance at 517 nm induced by antioxidants. The absorption maxi-
mum of a stable DPPH radical in ethanol was 517 nm. Because of the
reaction between antioxidant molecules and radicals, the absorbance
of the DPPH radical caused by antioxidants decreased, which results in
radical scavenging by hydrogen donation. This process is visually
noticeable as a change from purple to yellow. Hence, DPPH. is usually
Fe2+ + H2O2 → Fe3+ + OH− + OH•.
The Fe3+ ion also produces radicals from peroxides, although the
rate is tenfold less than that of the Fe2+ ion, which is the most powerful
pro-oxidant among the various types of metal ions [41]. Ferrous ion-
chelating activities of the compounds 4 with EDTA and α-tocopherol
are shown in Fig. 2. In this study, metal chelating capacity was signifi-
cant because it reduced the concentration of the catalyzing transition
metal. It was reported that chelating agents that form σ-bonds with a
metal are effective as secondary antioxidants because they reduce the
redox potential, thereby stabilizing the oxidized form of the metal ion
[42]. The data obtained from Fig. 2 reveal that the compounds, especially
Fig. 4. Overlapping chromatograms of 4a obtained by using different mobile phases (95:5, 90:10, 85:15, 80:20 MeOH/water (v/v)): Experimental conditions: flow rate = 1 mL min−1
detection wavelength = 260 nm, injection volume = 20 μL.
,

364
H. Yüksek et al. / Journal of Molecular Liquids 206 (2015) 359–366
Table 3
4d and 4h, demonstrate a marked capacity for iron binding, suggesting
that their action as peroxidation protectors may be related to their iron-
binding capacity. The metal-chelating effect of the compounds and stan-
dards decreased in the order of EDTA N α-tocopherol N 4h N 4d. On the
other hand, free iron is known to have low solubility and a chelated iron
complex has greater solubility in solution. Furthermore, the compound-
iron complex may also be active because it can participate in iron-
catalyzed reactions.
The parameters for the decomposition of compounds 4a–h.
Compounds
Ea (kJ/mol)
r2
H–M
C–R
H–M
C–R
4a
4b
4c
4d
4e
4f
133.2
139.5
136.8
143.5
149.3
151.9
159.5
157.3
139.2
141.6
135.3
146.8
152.3
153.6
161.5
160.8
0.998
0.997
0.999
0.995
0.996
0.996
0.997
0.995
0.999
0.998
0.997
0.997
0.995
0.999
0.998
0.996
4g
4h
3.3. Potentiometric titrations
To determine the pKa values of the compounds 4a–h, they were
titrated potentiometrically with TBAH in four non-aqueous solvents:
isopropyl alcohol, tert-butyl alcohol, acetone and N,N-dimethyl form-
amide (DMF). The mV values read in each titration were plotted against
the 0.05 M TBAH volumes (mL) added, and potentiometric titration
curves were obtained for all the cases. From the titration curves, the
half-neutralization potential (HNP) values were measured, and the
corresponding pKa values were calculated. The data obtained from the
potentiometric titrations were interpreted, and the effect of the C-3 sub-
stituent on the 4,5-dihydro-1H-1,2,4-triazol-5-one ring, as well as
solvent effects, were studied [15–23,43].
It is well known that the acidity of a compound depends on certain
factors, the most important of which are the solvent effect and the
molecular structure [15–23,43]. Table 1 and Fig. 3 show that the HNP
values and corresponding pKa values obtained from the potentiometric
titrations depend on the non-aqueous solvents used and the substitu-
ents at C-3 on the 4,5-dihydro-1H-1,2,4-triazol-5-one ring.
3.4. HPLC analysis for determination of log k′w values
In the reverse phase HPLC system, the lipophilic molecules tend to
be distributed in the lipophilic stationary phase instead of hydrophilic
mobile phase. The more hydrophobic the molecule, the more time it
will spend on the stationary phase and the higher the concentration of
organic solvent will be required to promote elution.
As an example, the potentiometric titration curves for 0.001 M solu-
tions of compounds 4b titrated with 0.05 M TBAH in isopropyl alcohol,
tert-butyl alcohol, DMF and acetone are shown in Fig. 3.
When the solvent dielectric permittivity is taken into consideration,
the acidity order can be given as follows: DMF (ε = 36.7) N acetone
(ε = 36) N isopropyl alcohol (ε = 19.4) N tert-butyl alcohol (ε =
12). As seen in Table 1, the acidity order for compound 4a is:
acetone N DMF, for compound 4b it is: DMF N acetone, for compound
4f it is: acetone N tert-butyl alcohol, and for compound 4g it is: iso-
propyl alcohol N DMF. Moreover, as seen in Table 1, for compounds
4a–4f and 4h in isopropyl alcohol, for compounds 4c, 4d, and 4f in
DMF, for compounds 4a–4e, 4g, and 4h in tert-butyl alcohol and for
compounds 4c, 4e, 4g, and 4h in acetone, the HNP values and the cor-
responding pKa values were not obtained.
When the acidity power of compounds in solvents was researched
considering the dielectric constant, HNP and pKa values in tert-butyl
alcohol and isopropyl alcohol were not assigned because the titration
curves were not obtained in amphiprotic solvents. Therefore, the acidity
order was not conducted. It was determined that compound 4b was
more acidic than acetone in the DMF environment and was appropriate
with the theoretical order when studying dipolar aprotic solvents.
The relationship between log k′ and methanol concentration in the
mobile phase is known in HPLC theory, and it is described with the for-
mula: log k′ = log k′w − Sφ, where kw represents the k value for a com-
pound if the pure water is used as eluent, S is the slope of the regression
curve, and φ is the volume percentage of methanol in the mobile phase
[44,45]. For each studied compound, a linear correlation was found be-
tween log k′ and φ, and the correlation coefficients were all N0.99. The
values of S and the extrapolated log k′w are given in Table 2.
Octanol–water partition coefficients are the most widely used mea-
sures of lipophilicity in modeling biological partition/distribution. The
partition coefficient can be estimated by a classical shake-flask method.
However, it has many disadvantages, such as requiring relatively large
amounts of solutes and solvents and it is time-consuming.
It has long been recognized that the retention of a compound in
reversed-phase liquid chromatography is governed by its lipophilicity/
hydrophobicity, and thus correlates with the octanol–water partition
coefficient [46].
Fig. 5. Overlapping chromatograms of 4a–h by using 85:15 MeOH/water (v/v) mixture as the mobile phase: Experimental conditions: flow rate = 1 mL min−1, detection wavelength =
260 nm, injection volume = 20 μL.

H. Yüksek et al. / Journal of Molecular Liquids 206 (2015) 359–366
365
Reversed phase chromatography involves the adsorption of hydro-
phobic molecules onto a hydrophobic solid support in a polar mobile
phase. Decreasing the mobile phase polarity by adding more organic
solvent reduces the hydrophobic interaction between the solute and
the solid support resulting in desorption. The more hydrophobic the
molecule, the more time it will spend on the solid support and the
higher the concentration of organic solvent will be required to promote
desorption. Fig. 4 is given as an example to clarify this application. The
higher methanol content in the mobile phase makes 4a elute from the
HPLC column easily. The k′ value of 4a for different methanol composi-
tions is directly related with its lipophilicity. If the log k′ values were
extrapolated from binary eluents to 100% aqueous solutions, one
could estimate the log kw values of the synthesized compounds. In
that way, the linear relationship between log k′ and methanol composi-
tion as described above could be used to measure the synthesized mol-
ecules' lipophilicity.
Fig. 5 presents overlapping chromatograms of eight compounds
(4a–h) using an 85:15 MeOH/water (v/v) mixture as the mobile
phase. As seen in Fig. 5, the most hydrophilic compound was 4a,
which has a \CH3 group, and the most hydrophobic compound was
4e, which has a \CH2C6H4CH3 group, as expected. It is well known
that adding one more carbon to the aliphatic chain of the substituent
improves the lipophilicity of a compound in a homologue series. There-
fore, 4c was more lipophilic than 4a and 4b. On the other hand, the elec-
tronegative substituent on the phenyl group, –OCH3, strongly reduced
the lipophilicity of 4f, causing it to elute at almost the same retention
time as 4c. It is known that at the molecular level, solubility is controlled
by the energy balance of intermolecular forces between solute–solute,
solvent–solvent and solute–solvent molecules. Recall from general
chemistry that intermolecular forces come in different strengths, rang-
ing from very weak induced dipole–induced dipole interactions to
much stronger dipole–dipole forces (including the important special
case, hydrogen bonding). The experimental data in our study strongly
suggest that modifying the substituents on a homologue series could
dramatically change the molecular structure and the lipophilicity of
the compounds [47,48], and in that way it could also change their solu-
bility in aqueous media and the passive diffusion through cell mem-
branes [49]. The solubility of compounds could be used also when
trying to purify (e.g., recrystallization and choice of solvent) or isolate
them from a multi-component reaction mixture (e.g., via extraction),
or when extracting a molecule from a matrix.
and Coats–Redfern (C–R) equations are close to each other for the com-
pounds. The values from the two models are in close agreement with
each other.
4. Conclusions
The identification of new compounds for protecting cells from
oxidative stress is a rapidly rising area of scientific interest. Even several
natural sources that provide active components have been used to syn-
thesize and obtain effective new antioxidative compounds. It is well-
known that 1,2,4-triazole derivatives present an antitumor activity on
many cancer types, such as leukemia, non-small cell lung, colon, mela-
noma, ovarian, renal, prostate, and breast cancers. Several in vitro stud-
ies were performed for this purpose in recent years. The anticancer
activity of 1,2,4-triazole derivatives might be associated with their anti-
oxidant activity. There have been many reports about the biological
activities and synthesis of 1,2,4-triazole and 4,5-dihydro-1H-1,2,4-
triazol-5-one derivatives. In addition, several studies on the synthesis
of N-arylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-one derivatives are
available in the literature. However, the physicochemical properties of
the synthesized substances and biological activity have rarely been
discussed. In this study, the synthesis of eight new compounds was per-
formed and they were characterized by using IR, 1H NMR, 13C NMR and
UV spectral data. In addition, the pKa and lipophilicity of these com-
pounds were determined by using potentiometric titration and HPLC,
respectively. Kinetic parameters were also investigated. The compounds
were determined to possess an antioxidant activity and could be recom-
mended for further clinical investigations because such compounds
have already been reported to possess antitumor and antibacterial ac-
tivities in earlier studies.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgments
This work was supported by the Scientific Research Projects Coor-
dination Unit of Kafkas University (Project Number: 2013-FEF-75).
The authors thank Dr. Zafer Ocak (for determination of pKa values)
and Dr. Mustafa Calapoglu (for antioxidant activities).
3.5. Kinetic analysis
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