InVitro antioxidant activities of new 4,5-dihydro-1H-1,2,4-triazol-5-ones having thiophene ring with their acidic properties

Article abstract of DOI:10.1155/2011/635459

Seven new 3-alkyl(aryl)-4-(2-thienymethylenamino)-4,5-dihydro- 1H-1,2,4-triazol-5-ones (2) were synthesized by the reactions of 3-alkyl(aryl)- 4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (1) with thiophene-2-carbaldehyde. In addition, N-acetyl derivatives of compounds 2d-2g were also prepared. The structures of eleven new compounds synthesized were determined by elemental analysis as well as IR, NMR and UV spectral data. In addition, compounds 2a-g and 3a, 3b, 3d-f were also screened for their antioxidant activities and 2a-g were potentiometrically titrated with tetrabutylammonium hydroxide (TBAH) in four nonaqueous solvents (isopropyl alcohol, t-butyl alcohol, acetonitrile and N,N-dimethyl formamide). Also half-neutralization potential values and the corresponding pKa values were determined in all cases. Copyright E-Journal of Chemistry 2004-2011.

Full text of DOI:10.1155/2011/635459

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
http://www.e-journals.net
2011, 8(4), 1734-1746
InVitro Antioxidant Activities of New
4,5-Dihydro-1H-1,2,4-triazol-5-ones having
Thiophene Ring with their Acidic Properties
H. YUKSEK, F. ISLAMOGLU*, O. GURSOY KOL,
S. BAHCECI§, M. BEKAR§ and M. AKSOY§
Department of Chemistry, Faculty of Science and Arts
Kafkas University, 36100 Kars, Turkey
*Department of Chemistry, Faculty of Science and Arts
Rize University, 53100 Rize, Turkey
§Fatih Education Faculty, Karadeniz Technical University
61355 Trabzon, Turkey
fatihislamoglu53@hotmail.com
Received 19 February 2011; Accepted 16 April 2011
Abstract: Seven new 3-alkyl(aryl)-4-(2-thienymethylenamino)-4,5-dihydro-
1H-1,2,4-triazol-5-ones (2) were synthesized by the reactions of 3-alkyl(aryl)-
4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (1) with thiophene-2-carbaldehyde.
In addition, N-acetyl derivatives of compounds 2d-2g were also prepared. The
structures of eleven new compounds synthesized were determined by
elemental analysis as well as IR, NMR and UV spectral data. In addition,
compounds 2a-g and 3a, 3b, 3d-f were also screened for their antioxidant
activities and 2a-g were potentiometrically titrated with tetrabutylammonium
hydroxide (TBAH) in four nonaqueous solvents (isopropyl alcohol, t-butyl
alcohol, acetonitrile and N,N-dimethyl formamide). Also half-neutralization
potential values and the corresponding pKa values were determined in all
cases.
Keywords: 4,5-Dihydro-1H-1,2,4-triazol-5-ones, Synthesis, Schiff bases, Acetylation, Acidity,
Potentiometric titrations
Introduction
Several articles, involving the synthesis of some N-arylidenamino-4,5-dihydro-1H-1,2,4-
triazol-5-one derivatives, have been published1-9. Also, the reactions of some 4,5-dihydro-
1H-1,2,4-triazol-5-one derivatives with acetic anhydride were investigated5-10. In addition,
1,2,4-triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives are reported to show a broad

1735
F. ISLAMOGLU et al.
spectrum of biological activities4,11-15. On the other hand, there have been a number of
systematic studies of the acidity in different media using different techniques6-30, but
unfortunately very few have dealt with triazoles. It is well known that two major factors
influence the acidity of a molecule31-34, namely, structural and solvent effects. In most
molecules there are two or more structural effects and it is usually very difficult to assess
how much each effect contributes to the acidity of a molecule. Moreover, it is sometimes
extremely difficult to differentiate between structural and solvent effects.
Furthermore, antioxidants are extensively studied for their capacity to protect organisms
and cells from damage induced by oxidative stress. Scientists in various disciplines have
become more interested in new compounds, either synthesized or obtained from natural
sources, that could provide active components to prevent or reduce the impact of oxidative
stress on cells35. Exogenous chemicals and endogenous metabolic processes in human body or
in food system might produce highly reactive free radicals, especially oxygen derived radicals,
which are capable of oxidizing biomolecules, resulting in cell death and issue damage.
Oxidative damages play a significant pathological role in human diseases. For example,
cancer, emphysema, cirrhosis, atherosclerosis and arthritis have all been correlated with
oxidative damage. Also, excessive generation of ROS (reactive oxygen species) induced by
various stimuli and which exceeds the antioxidant capacity of the organism leads to a variety
of pathophysiological processes such as inflammation, diabetes, genotoxicity and cancer36.
This paper describes the synthesis of a series of 3-alkyl(aryl)-4-(2-thienymethy-
lenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (2) from the reactions of 3-alkyl(aryl)-4-
amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (1) with thiophene-2-carbaldehyde. Besides, the
reactions of compounds 2a, 2b, 2d-g with acetic anhydride affording the corresponding
compounds 3a, 3b, 3d-g were investigated (Scheme 1). Furthermore, the antioxidant
activities of 13 new compounds was determined.
Scheme 1. Synthesis route of compounds 1 - 3
Experimental
Melting points were taken on a electrothermal digital melting point apparatus and are uncorrected.
IR spectra (wavenumbers in cm-1) were registered on a Perkin-Elmer 1600 FTIR spectrometer.
1H NMR spectra were recorded in deuterated dimethyl sulfoxide on a Varian 60A spectrometer
with TMS as internal standard. Chemical shifts are given in ppm (δ-scale). UV absorption spectra
(λmax in nm) were measured in 10 mm quartz cells between 200 and 400 nm using a Shimadzu UV-
1201 spectrophotometer. Elemental analyses were performed using a Carlo-Erba 1106 analyzer.
The starting compounds 1a-1g were prepared from the reactions of the corresponding ester
ethoxycarbonylhydrazones with hydrazine hydrate according to the literature10,37
.
General procedure for preparation of 3-alkyl(aryl)-4-(2-thienymethylenamino)-4,5-
dihydro-1H-1,2,4-triazol-5-ones (2)
3-Alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-one (1) (0.01 mol) was heated with
thiophene-2-carbaldehyde (1.12 g, 0.01 mol) at 160-165 °C (170-175 °C for compounds
2d-2g) for 1.5 h and cooled. Several recrystallizations of the residue from an appropriate
solvent gave pure compounds 2.

In Vitro Antioxidant Activities of New Thiophene Ring
1736
3-Methyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (2a)
Colourless crystals, yield 99%. M.p. 174 °C (EtOH). For C8H8N4OS (208.2) calculated: 46.14%
1
C, 3.87% H, 26.91% N; found: 45.65% C, 3.63% H, 27.70% N. H NMR (DMSO-d6): 2.30
(s, 3H, CH3); 7.10-7.34 (m, 1H, Ar-H); 7.60-7.90 (m, 2H, Ar-H); 9.82 (s, 1H, N=CH); 11.70
(s, 1H, NH). IR (KBr): 3140 (NH), 1698 (C=O), 1598, 1585 (C=N) cm-1. UV (ethanol), λmax (ε):
313 (11020), 264 (7200), 207 (5940) nm. MS: m/z 231 (M+23), 185, 169, 142, 115, 96 (100), 69, 60.
3-Ethyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (2b)
Colourless crystals, yield 98%. M.p. 157 °C (EtOH-H2O, 1 : 3). For C9H10N4OS (222.3)
1
calculated: 48.64% C, 4.54% H, 25.22% N; found: 48.49% C, 4.45% H, 25.39% N. H
NMR (DMSO-d6): 1.34 (t, 3H, CH3); 2.76 (q, 2H, CH2); 7.10-7.34 (m, 1H, Ar-H); 7.60-7.98
(m, 2H, Ar-H); 9.82 (s, 1H, N=CH); 11.70 (s, 1H, NH). IR (KBr): 3150 (NH), 1708 (C=O),
1600, 1590 (C=N) cm-1. UV (ethanol), λmax (ε): 332 (10490), 319 (11560), 280 (8850), 222
(8260) nm. MS: m/z 245 (M+23), 239, 231, 185, 169, 142, 96 (100), 69, 60.
3-n-Propyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (2c)
Colourless crystals, yield 98%. M.p. 158 °C (EtOH-H2O, 1 : 3). For C10H12N4OS (236.3)
calculated: 50.83% C, 5.12% H, 23.71% N; found: 49.29% C, 4.76% H, 23.88% N. 1H NMR
(DMSO-d6): 0.98 (t, 3H, CH3); 1.68 (sext, 2H, CH2); 2.64 (t, 2H, CH2); 7.08-7.28 (m, 1H, Ar-
H); 7.60-7.86 (m, 2H, Ar-H); 9.76 (s, 1 H, N=CH); 11.80 (s, 1H, NH). IR (KBr): 3192 (NH),
1718 (C=O), 1602, 1590 (C=N) cm-1. UV (ethanol), λmax (ε): 312 (11310, 264 (6910), 203
(6510) nm. MS: m/z 259 (M+23), 237 (M+1), 236 (M+), 234, 185, 169, 142, 96 (100), 93, 69.
3-Benzyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2d)
Colourless crystals, yield 97%. M.p. 188 °C (EtOH-H2O, 1 : 3). For C14H12N4OS (284.3)
1
calculated: 59.14% C, 4.26% H, 19.72% N; found: 59.00% C, 4.15% H, 19.62% N. H
NMR (DMSO-d6): 4.14 (s, 2H, CH2); 7.14-7.44 (m, 6H, Ar-H); 7.60-7.90 (m, 2H, Ar-H);
9.80 (s, 1H, N=CH); 11.90 (s, 1H, NH). IR (KBr): 3192 (NH), 1715 (C=O), 1605, 1590
(C=N), 755, 700 (monosubstituted benzenoid ring) cm-1. UV (ethanol), λmax (ε): 319
(10560), 305 (12900), 264 (9650), 210 (11690) nm. MS: m/z 307 (M+23), 285 (M+1), 284
(M+), 265, 234, 198, 185, 161, 142, 96 (100), 93, 69.
3-(p-Methylbenzyl)-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2e)
Colourless crystals, yield 89%. M.p. 151 °C (EtOH-H2O, 1 : 3). For C15H14N4OS (298.4)
1
calculated: 60.38% C, 4.73% H, 18.79% N; found: 59.89% C, 4.53% H, 18.07% N. H
NMR (DMSO-d6): 2.30 (s, 3H, CH3); 3.96 (s, 2H, CH2); 6.90-7.26 (m, 5H, Ar-H); 7.56-7.80
(m, 2H, Ar-H); 9.76 (s, 1H, N=CH); 11.80 (s, 1H, NH). IR (KBr): 3195 (NH), 1720 (C=O),
1605, 1595 (C=N), 820 (1,4-disubstituted benzenoid ring) cm-1. UV (ethanol), λmax (ε): 320
(8640), 281 (7060), 223 (10890) nm. MS: m/z 321 (M+23), 299 (M+1), 265, 234, 231, 198,
185, 169, 161, 142, 96 (100), 93, 69, 60.
3-(4-Chlorobenzyl)-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2f)
Colourless crystals, yield 94%. M.p. 192 °C (EtOH). For C14H11N4OSCl (318.8) calculated:
52.82% C, 3.49% H, 17.61% N; found: 52.93% C, 3.31% H, 17.48% N. 1H NMR (DMSO-d6):
4.04 (s, 2H, CH2); 7.08-7.28 (m, 1H, Ar-H); 7.34 (s, 4H, Ar-H); 7.56-7.86 (m, 2H, Ar-H); 9.76
(s, 1H, N=CH); 11.84 (s, 1H, NH). IR (KBr): 3195 (NH), 1715 (C=O), 1600, 1585 (C=N), 820
(1,4-disubstituted benzenoid ring) cm-1. UV (ethanol), λmax (ε): 311 (13090), 266 (10670), 220
(14420) nm. MS: m/z 321, 320 (M+2), 319 (100), 309, 268, 265, 254, 234, 142, 110, 93, 79, 60.

1737
F. ISLAMOGLU et al.
3-Phenyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2g)
Colourless crystals, yield 69%. M.p. 164 °C (benzene). For C13H10N4OS (270.3) calculated:
1
57.76% C, 3.73% H, 20.73% N; found: 57.64% C, 3.60% H, 20.55% N. H NMR (DMSO-
d6): 7.10-8.00 (m, 8H, Ar-H); 9.74 (s, 1H, N=CH); 12.16 (s, 1H, NH). IR (KBr): 3195 (NH),
1720 (C=O), 1595, 1570 (C=N), 762, 710 (monosubstituted benzenoid ring) cm-1. UV
(ethanol), λmax (ε): 325 (8450), 319 (10780), 305 (12990), 264 (17220), 225 (8450), 210
(11720) nm. MS: m/z 293 (M+23), 271 (M+1), 234, 198, 185, 169, 142, 96 (100), 93, 60.
General procedure for preparation of 1-Acetyl-3-alkyl(aryl)-4-(2-thienymethy-
lenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (3)
The corresponding compound 2 (0.01 mol) was refluxed with acetic anhydride (15 mL) for
0.5 h. After addition of absolute ethanol (50 mL), the mixture was refluxed for 1 h.
Evaporation of the resulting solution at 40-45 °C in vacuo and several recrystallizations of
the residue from an appropriate solvent gave pure compounds 3.
1-Acetyl-3-methyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3a)
1
Colourless crystals, yield 91%. M.p. 178 °C (ethanol). H NMR (DMSO-d6): 2.25 (s, 3H,
CH3); 2.44 (s, 3H, COCH3); 7.19 (t, 1H, Ar-H, J=4.76 Hz); 7.71 (d, 1H, Ar-H, J=3.66 Hz);
7.82 (d, 1H, Ar-H, J=5.12 Hz); 9.67 (s, 1H, N=CH). 13C NMR (DMSO-d6): 11.81 (CH3);
24.08 (COCH3); 129.06, 132.33, 135.45, 138.11 (ar-C); 147.02 (triazole-C3); 148.57
(N=CH); 151.52 (triazole-C5); 166.65 (C=O). IR (KBr): 1763 (C=O), 1613, 1587 (C=N) cm-1.
UV (ethanol), λmax (ε): 312 (16056), 260 (11280), 218 (17140) nm. MS: m/z 273 (M+23),
251 (M+1), 234, 231, 209, 147, 142, 120, 93, 74 (100), 69.
1-Acetyl-3-ethyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3b)
1
Colourless crystals, yield 79%. M.p. 179 °C (ethanol). H NMR (DMSO-d6): 1.20 (t, 3H,
CH3, J=7.32 Hz); 2.46 (s, 3H, COCH3); 2.65 (q, 2H, CH2, J=7.32 Hz); 7.20 (t, 1H, Ar-H,
J=4.39 Hz); 7.73 (d, 1H, Ar-H, J=3.66 Hz); 7.83 (d, 1H, Ar-H, J=5.12 Hz); 9.68 (s, 1H,
N=CH). 13C NMR (DMSO-d6): 10.03 (CH3); 19.29 (CH3); 24.12 (COCH3); 129.10, 132.34,
135.43, 138.20 (ar-C); 148.83 (triazole-C3); 150.53 (N=CH); 151.59 (triazole-C5); 166.63
(C=O). IR (KBr): 1769, 1687 (C=O), 1613, 1587 (C=N) cm-1. UV (ethanol), λmax (ε): 312
(17412), 260 (12684), 218 (7625) nm. MS: m/z 291, 223 (100), 217, 200, 120.
1-Acetyl-3-benzyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3d)
Colourless crystals, yield 95%. M.p. 167 °C (benzene-petroleum ether, 1 : 3). For
C16H14N4O2S (326.4) calculated: 58.88% C, 4.32% H, 17.17% N; found: 59.07% C, 4.27%
1
H, 16.65% N. H NMR (DMSO-d6): 2.58 (s, 3H, COCH3); 4.10 (s, 2H, CH2); 7.00-7.50
(m, 6H, Ar-H); 7.60-7.90 (m, 2H, Ar-H); 9.80 (s, 1H, N=CH). IR (KBr): 1753 (C=O), 1625,
1572 (C=N), 756, 696 (monosubstituted benzenoid ring) cm-1. UV (ethanol), λmax (ε): 314
(11730), 260 (9660), 209 (14790) nm. MS: m/z 327 (M+1), 308, 307, 285, 265, 231, 207,
185, 169, 147, 142, 96, 93, 74 (100), 69, 60.
1-Acetyl-3-(p-methylbenzyl)-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol
-5-one (3e)
Colourless crystals, yield 95%. M.p. 188 °C (EtOH). For C17H16N4O2S (340.4) calculated:
59.98% C, 4.74% H, 16.46% N; found: 59.50% C, 4.68% H, 16.02% N. 1H NMR (DMSO-d6):
2.34 (s, 3H, CH3); 2.62 (s, 3H, COCH3); 4.10 (s, 2H, CH2); 7.00-7.40 (m, 5H, Ar-H);
7.60-7.90 (m, 2H, Ar-H); 9.80 (s, 1H, N=CH). IR (KBr): 1734 (C=O), 1607, 1560 (C=N),

In Vitro Antioxidant Activities of New Thiophene Ring
1738
827 (1,4-disubstituted benzenoid ring) cm-1. UV (ethanol), λmax (ε): 312 (12018), 261
(9790), 207 (16210) nm. MS: m/z 340 (M+1), 321, 301, 299, 296, 282, 255, 239, 231, 209,
195, 185, 173, 169, 147, 145, 142 (100), 139, 120, 110.
1-Acetyl-3-(4-chlorobenzyl)-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol
-5-one (3f)
Colourless crystals, yield 95%. M.p. 200 °C (benzene-petroleum ether, 1 : 3). For
C16H13N4O2SCl (360.8) calculated: 53.26% C, 3.63% H, 15.53% N; found: 53.67% C,
1
3.60% H, 14.98% N. H NMR (DMSO-d6): 2.60 (s, 3H, COCH3); 4.16 (s, 2H, CH2); 7.08-
7.28 (m, 1H, Ar-H); 7.36 (s, 4H, Ar-H); 7.66-7.94 (m, 2H, Ar-H); 9.68 (s, 1H, N=CH). IR
(KBr): 1753 (C=O), 1625, 1565 (C=N), 820 (1,4-disubstituted benzenoid ring) cm-1. UV
(ethanol), λmax (ε): 312 (14320), 261 (11010), 207 (17740) nm. MS: m/z 352, 341, 321, 319,
279, 255, 226, 209, 195, 176, 147, 139, 125, 120 (100), 115.
1-Acetyl-3-phenyl-4-(2-thienymethylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3g)
Colourless crystals, yield 86%. M.p. 160 °C (EtOH). For C15H12N4O2S (312.4) calculated:
57.68% C, 3.87% H, 17.94% N; found: 57.90% C, 3.85% H, 17.55% N. 1H NMR (DMSO-d6):
2.60 (s, 3H, COCH3); 7.06-7.28 (m, 1H, Ar-H); 7.36-8.00 (m, 7H, Ar-H); 9.64 (s, 1H,
N=CH). IR (KBr): 1727 (C=O), 1600, 1570 (C=N), 762, 700 (monosubstituted benzenoid
ring) cm-1. UV (ethanol), λmax (ε): 313 (13 920), 262 (19 650), 206 (18 440) nm.
Antioxidant activity
Chemicals
Butylated hydroxytoluene (BHT) was purchased from E. Merck. 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) and trichloracetic acid
(TCA) were bought from Sigma (Sigma–Aldrich GmbH, Sternheim,Germany).
Reducing power
The reducing power of the synthesized compounds was determined according to the method
of Oyaizu38. Different concentrations of the samples (50-250 µg/mL) in DMSO (1 mL) were
mixed with phosphate buffer (2.5 mL, 0.2 M, pH = 6.6) and potassium ferricyanide (2.5 mL, 1%).
o
The mixture was incubated at 50 C for 20 min. After which a portion (2.5 mL) of
trichloroacetic acid (10%) was added to the mixture, which was then centrifuged for 10 min at
1000 x g. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and
FeCl3 (0.5 mL, 0.1%) and then the absorbance at 700 nm was measured in a spectrophometer.
Higher absorbance of the reaction mixture indicated greater reducing power.
Free radical scavenging activity
Free radical scavenging activity of compounds was measured by DPPH., using the method
of Blois39. 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 concentrations (50-250 µg/mL).
The mixture was shaken vigorously and allowed to stand at room temperature for 30 min.
Then the absorbance was measured at 517 nm in a spectrophometer. Lower absorbance of
the reaction mixture indicated 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 (R: 0.997):
Absorbance = 0.0003 x DPPH. – 0.0174
(1)

1739
F. ISLAMOGLU et al.
The capability to scavenge the DPPH radical was calculated using the following equation:
DPPH. scavenging effect (%) = (A0 – A1/A0) x 100
(2)
Where A0 is the absorbance of the control reaction and A1 is the absorbance in the
presence of the samples or standards.
Metal chelating activity
The chelation of ferrous ions by the synthesized compounds and standards were estimated
by the method of Dinis et al.40 Briefly, the synthesized compounds (50-250 µg/mL) were
added to a 2 mM solution of FeCl2 (0.05 mL). The reaction was initiated by the addition of 5 mM
ferrozine (0.2 mL) and 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 then measured at 562 nm in a spectrophotometer. All test and analyses were
run in triplicate and averaged. The percentage of inhibition of ferrozine-Fe2+ complex
formation was given by the formula: % Inhibition = (A0 – A1/A0) x 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.
Potentiometric titrations
Potentiometric titrations an Orion 720A model pH-ionmeter equipped with a combined pH
electrode (Ingold) and indicator electrode were used. A magnetic stirrer, a semi-micro burette and
a 25 mL beaker were also used in titrations. Before potentiometric titrations, the pH meter was
calibrated according to the instructions supplied by the manufactures of the pH meter. During the
titrations, the titrant was added in increments of 0.05 mL after each stable reading and mV values
were recorded. The necessary chemicals were supplied from Fluka and Merck. After
purifications, isopropyl alcohol was used to prepare 0.05 N tetra-butylammonium hydroxide. For
all potentiometric titrations, 0.05 N tetra-butylammonium hydroxide in isopropyl alcohol, which
was prepared from 0.1 N tetra-butylammonium hydroxide (TBAH) by dilution, was used. The
0.05 M solution of TBAH in isopropyl alcohol, which is widely used in the titration of acids, was
used as titrant. The half-neutralization potentials and the corresponding pKa values for all
compounds, obtained from the potentiometric titrations with 0.05 M tetrabutylammonium
hydroxide in isopropyl alcohol, t-butyl alcohol, N,N-dimethylformamide and acetonitrile. The
mV values, that were obtained in pH-meter, were recorded. The half-neutralization potential
(HNP) values and the corresponding pKa values of all compounds, obtained from the
potentiometric titrations with 0.05 M TBAH in isopropyl alcohol, t-butyl alcohol, acetonitrile and
N,N-dimethylformamide, are presented in Table 1. Finally, the half-neutralization potential
(HNP) values were determined by drawing the mL (TBAH)-mV graphic. From the titration
curves, the HNP values were measured and the corresponding pKa values were calculated.
Results and Discussion
In this study, the structures of seven new 3-alkyl(aryl)-4-(2-thienymethylenamino)-4,5-
dihydro-1H-1,2,4-triazol-5-ones (2) and four new 1-acetyl-3-alkyl(aryl)-4-(2-thieny-
methylenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (3) were identified by using elemental
1
analysis and IR, H NMR and UV spectral data, and the obtained spectral values were seen
as compatible with literature4-6,15
.
Antioxidant activity
The compounds 2a-g and 3a, 3b, 3d-f were screened for their in-vitro antioxidant activities.
Several methods are used to determine antioxidant activities. The methods used in this study
are discussed below.

In Vitro Antioxidant Activities of New Thiophene Ring
1740
Table 1. Half-neutralization potential (HNP) values and the corresponding pKa values of all
triazole derivatives in isopropyl alcohol, N,N-dimethyl formamide, t-butyl alcohol and
acetonitrile
N,N-Dimethyl-
formamide
Isopropyl alcohol
pKa HNP, mV
t-Butyl alcohol
pKa HNP, mV
Acetonitrile
pKa HNP, mV
pKa HNP, mV
a
b
c
13.03 0.13 -356.8 1.3 14.43 0.19 -439.9 2.3 14.15 0.23 -422.7 1.8 14.97 0.19 -471.1 2.2
13.30 0.17 -372.7 1.9 14.64 0.15 -452.1 1.9 14.60 0.16 -448.8 2.2 14.96 0.15 -471.7 1.7
13.22 0.14 -368.0 1.8 15.59 0.17 -507.3 2.1 14.92 0.11 -468.8 1.5 15.53 0.12 -504.4 1.9
12.84 0.16 -345.4 1.5 14.37 0.11 -436.2 1.8 14.24 0.15 -428.4 1.7 14.67 0.20 -453.7 1.4
d
e
12.88 0.21 -347.9 1.6 14.59 0.19 -448.4 1.7 15.13 0.18 -481.1 2.3 14.91 0.13 -468.3 1.6
f
12.59 0.18 -330.6 1.4 14.13 0.13 -420.9 1.4 14.08 0.20 -418.8 1.6 14.72 0.11 -456.3 1.7
12.57 0.15 -329.5 1.8 14.04 0.16 -416.5 2.0 14.28 0.14 -431.3 1.4 14.27 0.18 -430.1 1.5
g
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. The reducing powers 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 for
its potential antioxidant activity41. The antioxidant activity of a putative antioxidant has been
attributed to various mechanisms, among which are prevention chain initiation, binding of
transition metal ion catalyst, decomposition of peroxides, prevention of continued hydrogen
abstraction, reductive capacity and radical scavenging42. In this study, all of the amounts of
the compounds showed lower absorbance then 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, compounds did not show the reductive activities.
DPPH• radical scavenging activity
The scavenging of the stable DPPH radical model is a widely used method to evaluate
antioxidant activities in a relatively short time compared with other methods. The effect of
antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating
ability43. DPPH is a stable free radical and accepts an electron or hydrogen radical to
become a stable diamagnetic molecule44. The reduction capability of DPPH radicals was
determined by decrease in its absorbance at 517 nm induced by antioxidants. The absorption
maximum of a stable DPPH radical in ethanol was at 517 nm.
The decrease in absorbance of DPPH radical was caused by antioxidants, because of
reaction between antioxidant molecules and radical, progresses, which result in the
scavenging of the radical by hydrogen donation. It is visually noticeable as a discoloration
from purple to yellow. Hence, DPPH is usually used as a substrate to evaluate antioxidative
activity of antioxidants45. BHA and α-tocopherol were used as a reference to antioxidant
compounds. Scavenging effect values of compounds 2g, 3e, BHA and α-tocopherol at
different concentrations are given Figure 1. The newly synthesized compounds showed no
activity as a radical scavenger.
Ferrous ion chelating activity
The chelating effect towards ferrous ions by the compounds and standards was determined
according to the method of Dinis40. Ferrozine can quantitatively form complexes with Fe2+.

1741
F. ISLAMOGLU et al.
In the presence of chelating agents, the complex formation is disrupted with the result that
the red colour of the complex is decreased. Measurement of colour reduction therefore
allows estimation of the chelating activity of the coexisting chelator46. Transition metals
have pivotal role in the generation oxygen free radicals in living organism.
35
30
25
2g
20
3e
BHA
15
α-T.pherol
10
5
0
0
5
10
15
20
25
30
35
40
Concentration, µg/mL
Figure 1. Scavenging effect of compounds 2g, 3e, BHA and α-tocopherol at different
concentrations (12.5-25-37.5 µg/mL)
The ferric iron (Fe3+) is the relatively biologically inactive form of iron. However, it can be
reduced to the active Fe2+, depending on condition, particularly47 pH and oxidized back through
Fenton type reactions with the production of hydroxyl radical or Haber-Weiss reactions with
superoxide anions. The production of these radicals may lead to lipid peroxidation, protein
modification and DNA damage. Chelating agents may not activate metal ions and potentially
inhibit the metal-dependent processes48. Also, the production of highly active ROS such as O2.-,
H2O2 and OH. is also catalyzed by free iron though Haber-Weiss reactions:
O.2 + H2O2 → O2 + OH- + OH.
(1)
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 reactive free radicals via the Fenton reactions:
Fe2+ + H2O2 → Fe3+ + OH- + OH.
(2)
Fe3+ ion also produces radicals from peroxides, although the rate is tenfold less than that
of Fe2+ ion, which is the most powerful pro-oxidant among the various types of metal ions49.
Ferrous ion chelating activities of the compounds, BHT, BHA and α-tocopherol are shown
in Figures 2 and 3. 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 σ-bonds with a metal are effective as secondary antioxidants because they reduce the
redox potential thereby stabilizing the oxidized form of metal ion50.
The data obtained from Figures 2 and 3 reveal that the compounds, except 2e,
demonstrate a marked capacity for iron binding, suggesting that 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.

In Vitro Antioxidant Activities of New Thiophene Ring
1742
40
35
30
25
20
15
10
5
2a
2b
2c
2d
2e
2f
2g
BHT
BHA
α-T.pherol
0
0
5
10
15
20
25
30
35
40
Concentration, µg/mL
Figure 2. Metal chelating effect of different amount of the compounds 2a-g, BHT, BHA and
α-tocopherol on ferrous ions
45
40
35
30
25
3a
3b
3d
3e
3f
20
BHT
15
BHA
α-T.pherol
10
5
0
0
5
10
15
20
25
30
35
40
Concentration, µg/mL
Figure 3. Metal chelating effect of different amount of the compounds 3a, 3b, 3d-f, BHT,
BHA and α-tocopherol on ferrous ions
Potentiometric titrations
In this study, compounds 2a-2g were titrated potentiometrically with TBAH in isopropyl
alcohol, t-butyl alcohol, acetonitrile and N,N-dimethyl formamide. The mV values read in
each titration were drawn against TBAH volumes (mL) added and potentiometric titration
curves were formed for all the cases. From the titration curves, the HNP values were
measured and the corresponding pKa values were calculated. The half-neutralization
potential (HNP) values and the corresponding pKa values of all triazole derivatives,
obtained from the potentiometric titrations with 0.05 M TBAH in isopropyl alcohol, t-butyl
alcohol, acetonitrile and N,N-dimethyl formamide, are presented in Table 1. Also the derived
potentiometric titration curves were plotted (Figures 4 – 8 for 2a). The pH of the weak acids
are given by the following equation:
pH = pKa + log [A–]/[HA]
pH = pKa occurs when [A–] is equal to [HA] at the half-neutralization point. Therefore,
the pH values can be regarded as pKa at the half-neutralization points. When the dielectric

1743
F. ISLAMOGLU et al.
permittivity of solvents is taken into consideration, the acidic arrangement can be expected
as follows: N,N-dimethyl formamide (ε=36.7) > acetonitrile (ε=36.0) > isopropyl alcohol
(ε=19.4) > t-butyl alcohol (ε=12.0). The acidity of a compound depends on several factors.
The two most important factors are the solvent effect and molecular structure. Table 1 shows
that the half neutralization potential (HNP) values and the corresponding pKa values
obtained from potentiometric titrations depend on the type of non-aqueous solvents used and
molecular structure of the compound.
20
18
16
14
T
Isopropyl alcohol
N,N-Dimethyl formamide
t-Butyl alcohol
Acetonitrile
12
10
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
TBAH, mL
Figure 4. pH - mL (TBAH) potentiometric titration curves of 0.001 M solutions of
compound 2a titrated with 0.05 M TBAH in isopropyl alcohol, t-butyl alcohol, acetonitrile
and N,N-dimethyl formamide at 25 °C
-200
T
Isopropyl alcohol
N,N-Dimethyl formamide
t-Butyl alcohol
-300
Acetonitrile
-400
-500
-600
-700
-800
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
TBAH, mL
Figure 5. mV – mL (TBAH) potentiometric titration curves of 0.001 M solutions of
compound 2a titrated with 0.05 M TBAH in isopropyl alcohol, t-butyl alcohol, acetonitrile
and N,N-dimethyl formamide at 25 °C

In Vitro Antioxidant Activities of New Thiophene Ring
1744
3000
2500
2000
1500
1000
500
Isopropyl alcohol
N,N-Dimethyl formamide
t-Butyl alcohol
Acetonitrile
0
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
TBAH, mL
Figure 6. ∆E/∆V - mL (TBAH) potentiometric titration curves of 0.001 M solutions of
compound 2a titrated with 0.05 M TBAH in isopropyl alcohol, t-butyl alcohol, acetonitrile
and N,N-dimethyl formamide at 25 °C
60000
Isopropyl alcohol
N,N-Dimethyl formamide
t-Butyl alcohol
Acetonitrile
40000
20000
0
-20000
-40000
-60000
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
TBAH, mL
Figure 7. ∆2E/∆V2 - mL (TBAH) potentiometric titration curves of 0.001 M solutions of
compound 2a titrated with 0.05 M TBAH in isopropyl alcohol, t-butyl alcohol, acetonitrile
and N,N-dimethyl formamide at 25 °C
0,05
Isopropyl alcohol
N,N-Dimethyl formamide
t-Butyl alcohol
0,04
Acetonitrile
0,03
0,02
0,01
0,00
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
TBAH, mL
Figure 8. ∆V/∆E - mL (TBAH) potentiometric titration curves of 0.001 M solutions of
compound 2a titrated with 0.05 M TBAH in isopropyl alcohol, t-butyl alcohol, acetonitrile
and N,N-dimethyl formamide at 25 °C

1745
F. ISLAMOGLU et al.
As seen in Table 1, the acidic order for compounds a, b, c, d and f is: isopropyl alcohol
> t-butyl alcohol > N,N-dimethyl formamide > acetonitrile, for compounds e and g is:
isopropyl alcohol > N,N-dimethyl formamide > acetonitrile > t-butyl alcohol. In isopropyl
alcohol, a, b, c, d, e, f and g compounds show the strongest acidic properties. a, b, c, d and f
compounds show the weakest acidic properties in acetonitrile, e and g compounds show the
weakest acidic properties in t-butyl alcohol. This situation may be attributed to the hydrogen
bonding between the negative ions formed and the solvent molecules in the amphiprotic
neutral solvents. Autoprotolysis is an acid-base reaction between identical solvent molecules
is which some act as an acid and others as a base.
Consequently, the extent of an autoprotolysis reaction depends both on the intrinsic
acidity and the instrinsic basicity of the solvent. The importance of the autoprotolysis
constant in titrations lies in its effect on the completeness of a titration reaction.
Half-neutralization potential (HNP) values and corresponding pKa values obtained from
the potentiometric titrations rely on the non-aqueous solvents used and the substituents at
C-3, in triazole ring.
Conclusion
1,2,4-Triazoles have broad spectrum of biological activities. The synthesis and in vitro
antioxidant evaluation of new 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives are described.
All of the compounds demonstrate a marked capacity for iron binding. Acidity of a
compound depends on mainly two factors, i.e. solvent effect and molecular structure.
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