IR spectra were registered using potassium bromide disks on a Shimadzu 408 spectrometer. ¹HNMR and ¹³C-NMR spectra were recorded in deuterated dimethyl sulfoxide with TMS as internal standard on a Varian-Mercury spectrometer at 200 MHz and 50 MHz, respectively. UV-Vis absorption spectra were measured for ethanol solutions in 10 mm quartz cells between 200 and 400 nm using a Shimadzu-1201 spectrophotometer. Melting points were taken on an Electrothermal 9100 digital melting point apparatus. Combustion analyses were performed on a Carlo Erba 1106 Elemental Analyzer. Chemicals were supplied from Fluka and Merck. The starting compounds 1a–f were prepared according to literature [8,27]. Compounds 3 were obtained throught recently reported methods [10].

3-Alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-one (1) (0.01 mol) was heated with 1.86 g (0.01 mol) of p-nitrobenzoyl chloride at 150–160 ^ºC for 1.5 h and cooled. Recrystallization of the crude product from an appropriate solvent gave pure compound 2.

3-Methyl-4-(p-nitrobenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2a). White crystals, yield 84 %; m.p. 259–60 °C (EtOH/water, 1:2); Calculated for C₁₀H₉N₅O₄: 45.6% C, 3.4 % H, 26.6% N; found: 45.0% C, 3.3% H, 26.6% N; ¹H-NMR (200 MHz, DMSO-d ₆): δ; 2.48 (s, 3H, CH₃), 8.54 (d, 2H, Ar-H; J=8.6 Hz), 8.72 (d, 2H, Ar-H; J=8.2 Hz), 12.18 (s, 2H, 2NH). ¹³C-NMR (50 MHz, DMSO-d ₆): δ 10.79 (CH₃), 124.24 (2C), 129.70 (2C), 136.69, 145.45 (aromatic carbons), 150.20 (triazole C₃), 153.45 (triazole C₅), 164.97 (C=O); IR (KBr): 3395 (NH), 1745, 1687 (C=O), 1610 (C=N), 820 (1,4-disubstituted benzenoid ring) cm⁻¹; UV (ethanol) λ_max (ε, L mol⁻¹ cm⁻¹): 254 (10020), 214 (7920) nm.

3-Ethyl-4-(p-nitrobenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2b). White crystals, yield 84 %; m.p. 238–240 ^°C (EtOH-water, 1:2); Calculated for C₁₁H₁₁N₅O₄: 47.7% C, 4.0% H, 25.3% N; found: 48.2% C, 4.0% H, 25.5% N; ¹³C-NMR (50 MHz, DMSO-d ₆): δ 10.07 (CH₃), 18.22 (CH₂), 124.15 (2C), 129.64 (2C), 136.85, 149.60 (aromatic carbons), 150.30 (triazole C₃), 153.80 (triazole C₅), 165.10 (C=O); IR (KBr): 3300, 3100 (NH), 1720, 1685 (C=O), 1610 (C=N), 800 (1,4-disubstituted benzenoid ring) cm⁻¹; UV (ethanol) λ_max (ε, L mol⁻¹ cm⁻¹): 251 (10550), 213 (8590) nm.

3-Benzyl-4-(p-nitrobenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2c). White crystals, yield 84 %; m.p. 255–257 °C (EtOH-toluene, 1:3); Calculated for C₁₆H₁₃N₅O₄: 56.6% C, 3.9% H, 20.6% N; found: 56.4% C, 3.9% H, 20.1% N; ¹H-NMR (200 MHz, DMSO-d ₆): δ 3.89 (s, 2H, CH₂), 7.29 (s, 5H, Ar-H), 8.14 (d, 2H, Ar-H; J= 7.9 Hz), 8.40 (d, 2H, Ar-H; J= 7.9 Hz), 11.80 (s, 1H, NH), 12.01 (s, 1H, NH); ¹³C-NMR (50 MHz, DMSO-d ₆): δ 31.40 (CH₂), 124.06 (2C), 127.17, 128.77 (2C), 129.08 (2C), 129.59 (2C), 135.03, 136.69, 147.19 (aromatic carbons), 150.05 (triazole C₃), 153.10 (triazole C₅), 164.73 (C=O); IR (KBr): 3275, 3150 (NH), 1730, 1680 (C=O), 1610 (C=N), 810 (1,4-disubstituted benzenoid ring), 770, 710 (monosubstituted benzenoid ring) cm⁻¹; UV (ethanol) λ_max (ε, L mol⁻¹ cm⁻¹): 258 (11010), 214 (11010) nm.

3-p-Methylbenzyl-4-(p-nitrobenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2d). White crystals, yield 89 %; m.p. 238–239 ^°C (EtOH-water, 1:2); Calculated for C₁₇H₁₅N₅O₄: 57.8% C, 4.3% H, 19.8% N; found: 58.3% C, 4.1% H, 19.7% N; ¹H-NMR (200 MHz, DMSO-d ₆): δ 2.27 (s, 3H, CH₃), 3.80 (s, 2H, CH₂), 7.13 (s, 4H, Ar-H), 8.14 (s, 2H, Ar-H), 8.41 (s, 2H, Ar-H), 11.74 (s, 1H, NH), 11.94 (s, 1H, NH); ¹³C-NMR (50 MHz, DMSO-d ₆): δ 20.39 (CH₃), 30.40 (CH₂), 123.64 (2C), 128.46 (2C), 128.84 (2C), 129.10 (2C), 131.60, 135.90, 136.20, 146.85 (aromatic carbons), 149.70 (triazole C₃), 152.75 (triazole C₅), 164.30 (C=O); IR (KBr): 3300, 3150 (NH), 1730, 1680 (C=O), 1610 (C=N), 815 (1,4-disubstituted benzenoid ring) cm⁻¹; UV (ethanol) λ_max (ε, L mol⁻¹ cm⁻¹): 258 (13860), 214 (13860) nm.

3-p-Chlorobenzyl-4-(p-nitrobenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2e). White crystals, yield 88 %; m.p. 246–247 °C (EtOH-water, 1:2); Calculated for C₁₆H₁₂N₅O₄Cl: 51.4% C, 3.2% H, 18.7% N; found: 52.2% C, 3.2% H, 18.7% N; ¹H-NMR (200 MHz, DMSO-d ₆): δ 4.07 (s, 2H, CH₂), 7.45–7.54 (m, 4H, Ar-H), 8.31 (d, 2H, Ar-H; J= 8.6 Hz), 8.58 (d, 2H, Ar-H; J= 8.2 Hz), 11.94 (s, 1H, NH), 12.19 (s, 1H, NH); ¹³C-NMR (50 MHz, DMSO-d ₆): δ 30.60 (CH₂), 124.12 (2C), 128.73 (2C), 129.64 (2C), 131.01 (2C), 132.10, 134.20, 136.60, 146.80 (aromatic carbons), 150.20 (triazole C₃), 153.10 (triazole C₅), 164.80 (C=O); IR (KBr): 3275, 3150 (NH), 1725, 1680 (C=O), 1610 (C=N), 820 (1,4-disubstituted benzenoid ring) cm⁻¹; UV (ethanol) λ_max (ε, L mol⁻¹ cm⁻¹): 252 (11590), 213 (9380) nm.

3-Phenyl-4-(p-nitrobenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (2f). White crystals, yield 82 %; m.p. 247–248 °C (EtOH-water, 1:2); Calculated for C₁₅H₁₁N₅O₄: 55.4% C, 3.4% H, 21.5% N; found: 55.2% C, 3.5% H, 21.3% N; ¹H-NMR (200 MHz, DMSO-d ₆): λ 7.53–7.62 (m, 3H, Ar-H), 7.80–7.88 (m, 2H, Ar-H), 8.21 (d, 2H, Ar-H; J= 7.3 Hz), 8.44 (d, 2H, Ar-H; J= 7.9 Hz), 12.19 (s, 1H, NH), 12.48 (s, 1H, NH); ¹³C-NMR (50 MHz, DMSO-d ₆): λ 124.30 (2C), 126.91 (2C), 129.24 (2C), 129.46 (2C), 131.10, 131.90, 136.40, 146.10 (aromatic carbons), 150.25 (triazole C₃), 153.40 (triazole C₅), 164.80 (C=O); IR (KBr): 3275, 3225 (NH), 1745, 1695 (C=O), 1610 (C=N), 820 (1,4-disubstituted benzenoid ring), 775, 700 (monosubstituted benzenoid ring) cm⁻¹; UV (ethanol) λ_max (ε, L mol⁻¹ cm⁻¹): 263 (17500), 222 (8160) nm.

Free radical scavenging activity of compounds was measured by DPPH. using the method of Blois [29]. Briefly, a 0.1 mM solution of DPPH. in ethanol was prepared, and this solution (1 mL) was added to the solutions in DMSO (3 mL) at different concentrations (50–150 mg/L). 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):

The capability to scavenge the DPPH radical was calculated using the following equation:

Where A₀ is the absorbance of the control reaction and A₁ is the absorbance in the presence of the samples or standards.

The chelating ferrous ions by the synthesized compounds and standards were estimated by the method of Dinis et al. [30]. Briefly, the synthesized compounds (12.5–37.5 mg/L) were added to a solution of 2 mM 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 spectrophotometrically 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:

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.

A Jenway 3040-model ion analyzer was used for potentiometric titrations. An Ingold pH electrode was used. For each compound that would be titrated, the 0.001 M solution was separately prepared in each non-aqueous solvent. The 0.05 M solution of TBAH in isopropyl alcohol, which is widely used in the titration of acids, was used as titrant. The mV values, that were obtained in pH-meter, were recorded. Finally, the HNP values were determined by drawing the mL (TBAH)-mV graphic.

The compounds, for which calculations were carried out, are indicated in Tables 2–7. The numbering system is shown in Scheme 1 . All the structures were fully optimized with the Gaussian 03 program at the B3LYP/6-311G theoretical level [31]. After the optimization, ¹H and ¹³C chemical shifts were calculated with GIAO method [20,32–34], using corresponding TMS shielding calculated at the same theoretical level as the reference. All the computations were done using an IBM X-225 e-server computer. Linear correlation analyses were carried out using SigmaPlot program. The quality of each correlation was judged examining R, the Pearson correlation coefficient [35].

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 of its potential antioxidant activity [36]. The antioxidant activity of 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 scavenging [37]. 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 order words, compounds 2a–f and 3a–c,e,f did not show the ability of electron donor to scavenge free radicals.

The scavenging 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 ability [38]. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [39]. 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 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 antioxidants [40]. In this study, antiradical activities of compounds and standard antioxidants such as BHA and α-tocopherol were determined by using DPPH. method. All the compounds tested with this method showed lower absorbance than absorbance of the control reaction and higher absorbance than that of the standard antioxidant reactions. The data obtained in this study indicate that the newly synthesized compounds showed mild activities as a radical scavenger, indicating that it has moderate activities as hydrogen donors. Figure 1 and Figure 2 illustrate a dose-dependent DPPH· radical scavenging activity of compounds tested and standards.

The chelating effect towards ferrous ions by the compounds and standards was determined according to the method of Dinis [30]. Ferrozine can quantitatively form complexes with Fe²⁺. 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 chelator [41]. Transition metals have pivotal role in the generation oxygen free radicals in living organism. The ferric iron (Fe³⁺) is the relatively biologically inactive form of iron. However, it can be reduced to the active Fe²⁺, depending on condition, particularly pH [42] 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 processes [43]. Also, the production of highly active ROS such as O₂ ^.– , H₂O₂ and OH^. is also catalyzed by free iron though Haber-Weiss reactions:

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:

Also Fe³⁺ ion produces radicals from peroxides, although the rate is tenfold less than of Fe²⁺ ion, which is the most powerful pro-oxidant among various sort of metal ions [44].

Ferrous ion chelating activities of 2a, 2b and 2e, BHT and α-tocopherol are shown in Figure 3 . 2a showed better ferrous chelating activity compared to standards at a concentration of 12,5 mg/L. Compounds 2a and 2e revealed good chelating activities at a concentration of 37,5 mg/L. Compounds 2c, 2d and 2f did not show any chelating effect. The 3a, 3b, 3c, 3e and 3f compounds tested with this method exhibited 66.9, 5.4, 72.4, 12.9 and 23.5 % chelation of ferrous ion at the concentration of 12.5 mg/L, respectively (Figure 4 ). Otherwise, the percentages of metal chelating capacity of same concentration of BHT and α-tocopherol were found 51,8 and 12,5 %, respectively (Figure 3 and Figure 4 ). Metal chelating capacity was significant since it reduced the concentrations of the catalyzing transition metal. It was reported that chelating agents, which form σ-bonds with a metal, are effective as secondary antioxidants because they reduce the redox potential thereby stabilizing the oxidized form of metal ion [45]. The data obtained from Figure 3 and Figure 4 reveal that the compounds, except for 2c, 2d and 2f, demonstrate a marked capacity for iron binding at the concentration-dependent manner, suggesting that their action as peroxidation protector may be related to its iron binding capacity. Moreover, free iron is known to have low solubility and a chelated iron complex has greater solubility in solution, which can be contributed solely from the ligand. Furthermore, the compound-iron complex may also be active, since it can participate to iron-catalyzed reactions.

In this study, the antioxidant activities of compounds varied with the tree test models. Our results revealed that all of the tested compounds showed mild antiradical activity and the compounds, except for 2c, 2d and 2f, revealed good chelating activities. In conclusion the data here reported could be of the possible interest because of their activities of radical scavenging and metal chelating could prevent redox cycling.

As a separate study, newly synthesized 2a–f type compounds were titrated potentiometrically with tetrabutylammonium hydroxide (TBAH) in four non-aqueous solvents such as acetone (ε=20.6), isopropyl alcohol (ε=19.4), tert-butyl alcohol (ε=12.0) and N,N-dimethylformamide (ε=36.7). The mV values read in each titration were drawn against 0.05 M TBAH volumes (mL) added, and thus potentiometric titration curves were formed for all the cases. From these curves, the HNP values were measured, and the corresponding pK _a values were calculated.

As an example, the potentiometric titration curves for 0.001 M compounds 2a–f solutions titrated with 0.05 M TBAH in acetone are shown Figure 5 .

The HNP values and the corresponding pK _a values for new compounds 2a–f, obtained from the potentiometric titrations with 0.05 M TBAH in acetone, isopropyl alcohol, tert-butyl alcohol and N,Ndimethylformamide, are presented in Table 1.

When the dielectric permittivity of solvents is taken into consideration, the acidic arrangement can be expected as follows: N,N-dimethylformamide (ε=36.7) > acetone (ε=20.6) > isopropyl alcohol (ε=19.4) > tert-butyl alcohol (ε=12.0). As seen in Table 1 , the acidic arrangement for compounds 2a, 2e and 2f is: isopropyl alcohol > tert-butyl alcohol > N,N-dimethylformamide > acetone, and for compounds 2b and 2d, it is : isopropyl alcohol > N,N-dimethylformamide > tert-butyl alcohol > acetone while the arrangement for compound 2c is : isopropyl alcohol > acetone > N,N-dimethylformamide > tert-butyl alcohol. In isopropyl alcohol, all these compounds show the strongest acidic properties, but they show the weakest acidic properties in acetone (for compound 2c in tert-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.

The degree to which a pure solvent ionizes was represented by its autoprotolysis constant, K_HS.

For the above reaction the constant is defined by

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 [46]. The exchange of the pK _a values with autoprotolysis constant and dielectric constant are given in Figure 6 .

As it is well known, the acidity of a compound depends on some factors. The two most important factors are the solvent effect and molecular structure [9–18]. Table 1 shows that the HNP values and corresponding pK _a values obtained from the potentiometric titrations depend on the non-aqueous solvents used and the substituents at C-3, in 4,5-dihydro-1H-1,2,4-triazol-5-one ring.

The experimental and theoretical ¹H and ¹³C chemical shifts, along with the error for each compound, are presented in Tables 2–7. A least-squares fit (theoretical calculation values/experimental calculation values) of all data shows a strong linear relationship with a R value of 0.999. This relationship is also reflected in the results for the individual compound, in which R values are 0.999, 0.976 (2a); 0.999 (2b); 0.999, 0.987 (2c); 0.999, 0.979 (2d); 0.999, 0.986 (2e); 0.999, 0.998 (2f). The overall standard error of estimate is 3.17 with the 6-311G basis set. The following correlations δ_calc = aδ_exp+ b were obtained: 2a (¹³C), SE (standard error) = 3.27, a = 1.04, b = 1.58; 2a (¹H), SE = 1.63, a = 0.60, b = 1.59; 2b (¹³C), SE = 2.94, a = 1.04, b = 1.65; 2c (¹³C), SE = 3.70, a = 1.03, b = 2.89; 2c (¹H), SE = 1.25, a = 0.41, b = 3.83; 2d (¹³C), SE = 4.23, a = 1.04, b = 2.96; 2d (¹H), SE = 1.40, a = 0.62, b = 1.99; 2e (¹³C), SE = 4.97, a = 1.04, b = 2.72; 2e (¹H), SE = 1.28, a = 0.40, b = 3.78; 2f (¹³C), SE = 2.83, a = 1.18, b = −1.68; 2f (¹H), SE = 0.49, a = −0.16, b = 9.20.

A linear correlation between theoretical and experimental carbon and proton chemical shifts was observed according to a, b and R values. A good quantitative agreement within 3.40–8.00 ppm in Tables 2–7 are observed for the aromatic carbons of these compounds. As can be seen at Tables 2–7, the errors range from 12.816 to 13.937 ppm for C-9 and C-14 for compound 2a due to attached nitrogen atoms. With the exception of the N-H protons, DFT method is in good agreement with the ¹H spectrum of compounds 2a–f. There has occurred an important difference between experimental and theoretical results more than it is expected. The reason why such a result has come into being is that the N-H hydrogen in 4,5-dihydro-1H-1,2,4-triazol-5-one ring has acidic properties, so that some 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives were titrated potentiometrically with TBAH in different non-aqueous solvents, and the pK _a values were found between 8.69 and 16.75 [4,9–18]. Thus, in the ¹H-NMR spectra of the 1,2,4-triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives, the signals of N-H protons were observed approximately δ 11 and 12 ppm [8–13,15–18,23,24,47,48]. This situation can be attributed to the resonance of the negatively formed ions (Scheme 2). Because of amide N-H proton, the errors range from 4.57 to 5.41 ppm for H-18 in compounds 2a, 2c–f were determined.