# Synthesis and In Vitro Antioxidant Evaluation of New 1,3,5-Tri-{2-methoxy-4-[(4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene Derivatives

Authors: H. Yuksek; O. Akyildirim; O. Gursoy Kol
Journal: Journal of Chemistry
 (2013)

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## Abstract

Nine new 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives were synthesized and characterized by elemental analyses and IR, 1H-NMR, 13C-NMR and UV spectral data. The synthesized compounds were analyzed for their in vitro potential antioxidant activities in three different methods. Those antioxidant activities were compared to standard antioxidants such as BHA, BHT and α-tocopherol. Compounds 4e, 5a and 5d showed best activity for iron binding. In addition, the compounds 4 were titrated potentiometrically with tetrabutylammonium hydroxide (TBAH) in four non-aqueous solvents (isopropyl alcohol, tert-butyl alcohol, acetone and N,N-dimethyl formamide). Thus, the half-neutralization potential values and the corresponding pKa values were determined in all cases.


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## Body

## 1. Introduction

1,2,4-Triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives are reported to possess a broad spectrum of biological activities such as antibacterial, antifungal, anti-inflammatory, antioxidant, and anticancer [1–9]. In addition, several articles reporting the synthesis of some N-arylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-one derivatives have been published [7, 8, 10–13]. The acetylation of 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives have also been reported [12–14].Furthermore, antioxidants have extensively been studied for their capacity to protect organism and cell from damage that are induced by oxidative stress. Scientists have become more interested in new compounds; they have either synthesized or obtained from natural sources that could provide active components to prevent or reduce the impact of oxidative stress on cell [15]. 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 significantly play a pathological role in human diseases. Cancer, emphysema, cirrhosis, atherosclerosis, and arthritis have all been correlated with oxidative damage. Also, excessive generation of ROS induced by various stimuli which exceeds the antioxidant capacity of the organism leads to a variety of pathophysiological processes such as inflammation, diabetes, genotoxicity and cancer [16]. In the present study, due to a wide range of applications to find their the possible radical scavenging and antioxidant activity, the newly synthesized compounds were investigated 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 nonaqueous solvents, and the pKa values of the compounds were determined [7, 8, 10, 12, 13, 17, 18].


## 2. Experimental

Chemical reagents used in this study were purchased from Merck AG, Aldrich, and Fluka. The starting materials3a–gwere prepared from the reactions of the corresponding ester ethoxycarbonylhydrazones 2a–gwith an aqueous solution of hydrazine hydrate as described in the literature [14, 19]. Melting points were determined in open glass capillaries using an electrothermal digital melting point apparatus and are uncorrected. The IR spectra were recorded on a Perkin-Elmer Instruments Spectrum One FT-IR spectrometer. 1H and 13C NMR spectra were recorded in deuterated dimethyl sulfoxide with TMS as internal standard using a Varian Mercury spectrometer at 200 MHz and 50 MHz, respectively. UV absorption spectra were measured in 10 mm quartz cells between 200 and 400 nm using a Schimadzu-1201 UV/VIS spectrometer. Extinction coefficients (ε) are expressed in L·mol-1·cm-1. Elemental analyses were carried out on an Leco 932 Elemental Combustion System (CHNS-O) for C, H, and N.


### 2.1. General Procedure for the Synthesis of Compounds4

3-Methoxy-4-hydroxybenzaldehyde (0.03 mol) dissolved in ethyl acetate (100 mL) was treated with 1,3,5-benzenetricarbonyl chloride (0.01 mol), and to this solution was slowly added triethylamine (0.03 mol) with stirring at 0–5°C. Stirring was continued for 2 h, and then the mixture was refluxed for 3 h and filtered. The filtrate was evaporatedin vacuo, and the crude product was washed with water and recrystallized from ethanol to afford compound 1, mp. 137°C; IR (KBr) (υ, cm−1): 2850 and 2739 (CHO); 1748, 1700 (C=O); 1274 (COO). 1H NMR (DMSO-d6): δ 3.87 (s, 9H, 3OCH3), 7.56–7.64 (m, 9H, ArH), 9.01 (s, 3H, ArH), 9.99 (s, 3H, CHO). 13C NMR (DMSO-d6): δ 56.06 (3OCH3), [111.90 (2C), 123.52 (3C), 123.63 (3C), 130.21 (4C), 135.46 (4C), 143.57 (4C), 151.26 (4C)] (Ar-C), 161.63 (3COO), 191.98 (3CHO). UV λmax(ε): 304 (9298), 256 (26386), 222 (39042), 212 (35722) nm. The corresponding compound 3 (0.003 mol) was dissolved in acetic acid (15 mL) and treated with 1,3,5-tri-(2-methoxy-4-formylphenoxycarbonyl)-benzene 1 (0.001 mol). The mixture was refluxed for 1.5 h and then evaporated at 50–55°C in vacuo. Several recrystallizations of the residue from AcOH-H2O (1 : 3) gave pure compounds 1,3,5-tri-{2-methoxy-4-[(3-alkyl/aryl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-benzene4as colorless crystals.


#### 2.1.1. 1,3,5-Tri-{2-methoxy-4-[(3-methyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4a)

Yield 0.67 g (74%). mp. 174°C. IR (KBr): 3223 (NH); 1751, 1715, 1705 (C=O); 1602 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 2.29 (s, 9H, 3CH3), 3.86 (s, 9H, 3OCH3), 7.45–7.68 (m, 9H, Ar-H), 9.02 (s, 3H, Ar-H), 9.75 (s, 3H, 3N=CH), 11.86 (s, 3H, 3NH). UV λmax(ε): 310 (41762), 262 (37725), 222 (64713) nm.


#### 2.1.2. 1,3,5-Tri-{2-methoxy-4-[(3-ethyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4b)

Yield 0.72 g (76%). mp. 319°C. IR (KBr): 3220 (NH); 1750, 1703 (C=O); 1600 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 1.18 (t, 9H, 3CH3,J=7.52 Hz), 2.70 (q, 6H, 3CH2,J=7.52 Hz), 3.88 (s, 9H, 3OCH3), 7.34–7.69 (m, 9H, Ar-H), 9.02 (s, 3H, Ar-H), 9.75 (s, 3H, 3N=CH), 11.89 (s, 3H, 3NH). UV λmax(ε): 310 (22888), 260 (27033), 224 (55717) nm.


#### 2.1.3. 1,3,5-Tri-{2-methoxy-4-[(3-n-propyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4c)

Yield 0.76 g (77%). mp. 168°C. IR (KBr): 3216 (NH); 1751, 1707 (C=O); 1598 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 0.94 (t, 9H, 3CH3,J=7.52 Hz), 1.68 (sext., 6H, 3CH2,J=7.52 Hz), 3.84 (s, 9H, 3OCH3), 2.64 (q, 6H, 3CH2,J=7.52 Hz), 7.41–7.66 (m, 9H, Ar-H), 9.00 (s, 3H, Ar-H), 9.74 (s, 3H, 3N=CH), 11,89 (s, 3H, 3NH). UV λmax(ε): 310 (23732), 260 (26373), 218 (52676) nm.


#### 2.1.4. 1,3,5-Tri-{2-methoxy-4-[(3-benzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4d)

Yield 1.03 g (91%). mp. 161°C. IR (KBr): 3214 (NH); 1750, 1718 (C=O); 1599 (C=N); 1272 (COO); 760 and 706 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.85 (s, 9H, 3OCH3), 4.07 (s, 6H, 3CH2), 7.20–7.68 (m, 24H, Ar-H), 9.02 (s, 3H, Ar-H), 9.70 (s, 3H, 3N=CH), 12.03 (s, 3H, 3NH). UV λmax(ε): 310 (13919), 258 (17758), 216 (45657) nm.


#### 2.1.5. 1,3,5-Tri-{2-methoxy-4-[(3-p-methylbenzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4e)

Yield 1.15 g (98%). mp. 216°C. IR (KBr): 3219 (NH); 1751, 1705 (C=O); 1600 (C=N); 1272 (COO); 829 (1,4-disubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 2.21 (s, 9H, 3CH3), 3.84 (s, 9H, 3OCH3), 4.00 (s, 6H, 3CH2), 7.06–7.65 (m, 21H, Ar-H), 9.02 (s, 3H, Ar-H), 9.69 (s, 3H, 3N=CH), 12.04 (s, 3H, 3NH). 13C NMR (DMSO-d6): δ 20.54 (3PhCH3), 30.83 (3CH2Ph), 56.07 (3OCH3), 111.93, 120.94 (arom-C), 123.50 (2C), 123.59 (2C), 128.57 (6C), 128.99 (6C), 130.35 (arom-C), 130.70 (3C), 132.68, 133.03 (arom-C), 135.46 (3C), 135.79 (3C), 141.13 (3C), 143.60 (3C), 146.36 (3C), 151.19 (3C), 150.95 (3triazole C3), 151.31 (N=CH), 151.91 (3triazole C5), 161.69 (3COO). UV λmax(ε): 300 (10113), 258 (13837), 226 (66208), 216 (61806) nm. Anal. Calcd. for C63H54N12O12 (1171.19): C, 64.61; H, 4.65; N, 14.35. Found: C, 64.85; H, 6.22; N, 13.29.


#### 2.1.6. 1,3,5-Tri-{2-methoxy-4-[(3-p-chlorobenzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4f)

Yield 0.89 g (72%). mp. 230°C. IR (KBr): 3210 (NH); 1750, 1717 (C=O); 1598 (C=N); 1271 (COO); 824 (1,4-disubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.88 (s, 9H, 3OCH3), 4.09 (s, 6H, 3CH2), 7.35–7.56 (m, 24H, Ar-H), 9.02 (s, 3H, Ar-H), 9.71 (s, 3H, 3N=CH), 12.04 (s, 3H, 3NH). UV λmax(ε): 310 (30831), 258 (47779), 222 (73777), 208 (58290) nm.


#### 2.1.7. 1,3,5-Tri-{2-methoxy-4-[(3-phenyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4g)

Yield 1.06 g (98%). mp. 243°C. IR (KBr): 3210 (NH); 1750, 1716 (C=O); 1602, 1585 (C=N); 1268 (COO); 768 and 693 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.84 (s, 9H, 3OCH3), 7.50–7.64 (m, 18H, Ar-H), 7.90–7.94 (m, 6H, Ar-H), 9.02 (s, 3H, Ar-H), 9.72 (s, 3H, 3N=CH), 12.41 (s, 3H, 3NH). UV λmax(ε): 308 (24520), 258 (44728), 234 (67930), 216 (64853) nm.


### 2.2. General Procedure for the Synthesis of Compound5

The corresponding compound4 (0.001 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 40–45°C in vacuo and several recrystallizations of the residue from EtOH gave pure compounds 5 as colorless needles.


#### 2.2.1. 1,3,5-Tri-{2-methoxy-4-[(1-acetyl-3-methyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene (5a)

Yield 0.66 g (64%). mp. 207°C. IR (KBr): 1728 (C=O); 1623, 1582 (C=N); 1268 (COO)cm-1. 1H NMR (DMSO-d6): δ 2.36 (s, 9H, 3CH3), 2.50 (s, 9H, 3COCH3), 3.85 (s, 9H, 3OCH3), 7.50–7.64 (m, 9H, Ar-H), 8.85 (s, 3H, Ar-H), 9.59 (s, 3H, 3N=CH). UV λmax(ε): 310 (20060), 258 (20237), 220 (63380), 216 (58300) nm.


#### 2.2.2. 1,3,5-Tri-{2-methoxy-4-[(1-acetyl-3-benzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene (5d)

Yield 0.88 g (71%). mp. 182°C. IR (KBr): 1746 (C=O); 1602, 1590 (C=N); 1217 (COO); 746 and 710 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 2.50 (s, 9H, 3COCH3), 3.85 (s, 9H, 3OCH3), 4.16 (s, 6H, 3CH2), 7.23–7.58 (m, 24H, Ar-H), 9.01 (s, 3H, Ar-H), 9.58 (s, 3H, 3N=CH). UV λmax(ε): 310 (45812), 296 (45995), 258 (47147), 230 (83429), 224 (81387) nm.


### 2.3. Antioxidant Activity: Chemicals

Butylated hydroxytoluene (BHT) was obtained from E. Merck (Merck KGaA, 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), and trichloroacetic acid (TCA) were obtained from Sigma (Sigma-Aldrich GmbH, Steinheim, Germany).


### 2.4. Reducing Power

The reducing power of the synthesized compounds was determined according to the method of Oyaizu [20]. 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%). 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 ×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.


### 2.5. Free Radical Scavenging Activity

Free radical scavenging activity of compounds was measured by DPPH•, using the method of Blois [21]. 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):
(1)Absorbance=0.0003×DPPH•-0.0174.
The capability to scavenge the DPPH radical was calculated using the following equation:
(2)DPPH•scavengingeffect(%)=(A0-A1A0)×100,
where A0 is the absorbance of the control reaction, and A1 is the absorbance in the presence of the samples or standards.


### 2.6. Metal Chelating Activity

The chelation of ferrous ions by the synthesized compounds and standards were estimated by the method of Dinis et al. [22]. 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 then the mixture was shaken vigorously and left standing at the 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+ 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.


### 2.7. Potentiometric Titrations

A Jenco model ion analyzer and an Ingold pH electrode were used for potentiometric titrations. For each compound that was 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 graph.


## 2.1. General Procedure for the Synthesis of Compounds4

3-Methoxy-4-hydroxybenzaldehyde (0.03 mol) dissolved in ethyl acetate (100 mL) was treated with 1,3,5-benzenetricarbonyl chloride (0.01 mol), and to this solution was slowly added triethylamine (0.03 mol) with stirring at 0–5°C. Stirring was continued for 2 h, and then the mixture was refluxed for 3 h and filtered. The filtrate was evaporatedin vacuo, and the crude product was washed with water and recrystallized from ethanol to afford compound 1, mp. 137°C; IR (KBr) (υ, cm−1): 2850 and 2739 (CHO); 1748, 1700 (C=O); 1274 (COO). 1H NMR (DMSO-d6): δ 3.87 (s, 9H, 3OCH3), 7.56–7.64 (m, 9H, ArH), 9.01 (s, 3H, ArH), 9.99 (s, 3H, CHO). 13C NMR (DMSO-d6): δ 56.06 (3OCH3), [111.90 (2C), 123.52 (3C), 123.63 (3C), 130.21 (4C), 135.46 (4C), 143.57 (4C), 151.26 (4C)] (Ar-C), 161.63 (3COO), 191.98 (3CHO). UV λmax(ε): 304 (9298), 256 (26386), 222 (39042), 212 (35722) nm. The corresponding compound 3 (0.003 mol) was dissolved in acetic acid (15 mL) and treated with 1,3,5-tri-(2-methoxy-4-formylphenoxycarbonyl)-benzene 1 (0.001 mol). The mixture was refluxed for 1.5 h and then evaporated at 50–55°C in vacuo. Several recrystallizations of the residue from AcOH-H2O (1 : 3) gave pure compounds 1,3,5-tri-{2-methoxy-4-[(3-alkyl/aryl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-benzene4as colorless crystals.


### 2.1.1. 1,3,5-Tri-{2-methoxy-4-[(3-methyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4a)

Yield 0.67 g (74%). mp. 174°C. IR (KBr): 3223 (NH); 1751, 1715, 1705 (C=O); 1602 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 2.29 (s, 9H, 3CH3), 3.86 (s, 9H, 3OCH3), 7.45–7.68 (m, 9H, Ar-H), 9.02 (s, 3H, Ar-H), 9.75 (s, 3H, 3N=CH), 11.86 (s, 3H, 3NH). UV λmax(ε): 310 (41762), 262 (37725), 222 (64713) nm.


### 2.1.2. 1,3,5-Tri-{2-methoxy-4-[(3-ethyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4b)

Yield 0.72 g (76%). mp. 319°C. IR (KBr): 3220 (NH); 1750, 1703 (C=O); 1600 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 1.18 (t, 9H, 3CH3,J=7.52 Hz), 2.70 (q, 6H, 3CH2,J=7.52 Hz), 3.88 (s, 9H, 3OCH3), 7.34–7.69 (m, 9H, Ar-H), 9.02 (s, 3H, Ar-H), 9.75 (s, 3H, 3N=CH), 11.89 (s, 3H, 3NH). UV λmax(ε): 310 (22888), 260 (27033), 224 (55717) nm.


### 2.1.3. 1,3,5-Tri-{2-methoxy-4-[(3-n-propyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4c)

Yield 0.76 g (77%). mp. 168°C. IR (KBr): 3216 (NH); 1751, 1707 (C=O); 1598 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 0.94 (t, 9H, 3CH3,J=7.52 Hz), 1.68 (sext., 6H, 3CH2,J=7.52 Hz), 3.84 (s, 9H, 3OCH3), 2.64 (q, 6H, 3CH2,J=7.52 Hz), 7.41–7.66 (m, 9H, Ar-H), 9.00 (s, 3H, Ar-H), 9.74 (s, 3H, 3N=CH), 11,89 (s, 3H, 3NH). UV λmax(ε): 310 (23732), 260 (26373), 218 (52676) nm.


### 2.1.4. 1,3,5-Tri-{2-methoxy-4-[(3-benzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4d)

Yield 1.03 g (91%). mp. 161°C. IR (KBr): 3214 (NH); 1750, 1718 (C=O); 1599 (C=N); 1272 (COO); 760 and 706 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.85 (s, 9H, 3OCH3), 4.07 (s, 6H, 3CH2), 7.20–7.68 (m, 24H, Ar-H), 9.02 (s, 3H, Ar-H), 9.70 (s, 3H, 3N=CH), 12.03 (s, 3H, 3NH). UV λmax(ε): 310 (13919), 258 (17758), 216 (45657) nm.


### 2.1.5. 1,3,5-Tri-{2-methoxy-4-[(3-p-methylbenzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4e)

Yield 1.15 g (98%). mp. 216°C. IR (KBr): 3219 (NH); 1751, 1705 (C=O); 1600 (C=N); 1272 (COO); 829 (1,4-disubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 2.21 (s, 9H, 3CH3), 3.84 (s, 9H, 3OCH3), 4.00 (s, 6H, 3CH2), 7.06–7.65 (m, 21H, Ar-H), 9.02 (s, 3H, Ar-H), 9.69 (s, 3H, 3N=CH), 12.04 (s, 3H, 3NH). 13C NMR (DMSO-d6): δ 20.54 (3PhCH3), 30.83 (3CH2Ph), 56.07 (3OCH3), 111.93, 120.94 (arom-C), 123.50 (2C), 123.59 (2C), 128.57 (6C), 128.99 (6C), 130.35 (arom-C), 130.70 (3C), 132.68, 133.03 (arom-C), 135.46 (3C), 135.79 (3C), 141.13 (3C), 143.60 (3C), 146.36 (3C), 151.19 (3C), 150.95 (3triazole C3), 151.31 (N=CH), 151.91 (3triazole C5), 161.69 (3COO). UV λmax(ε): 300 (10113), 258 (13837), 226 (66208), 216 (61806) nm. Anal. Calcd. for C63H54N12O12 (1171.19): C, 64.61; H, 4.65; N, 14.35. Found: C, 64.85; H, 6.22; N, 13.29.


### 2.1.6. 1,3,5-Tri-{2-methoxy-4-[(3-p-chlorobenzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4f)

Yield 0.89 g (72%). mp. 230°C. IR (KBr): 3210 (NH); 1750, 1717 (C=O); 1598 (C=N); 1271 (COO); 824 (1,4-disubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.88 (s, 9H, 3OCH3), 4.09 (s, 6H, 3CH2), 7.35–7.56 (m, 24H, Ar-H), 9.02 (s, 3H, Ar-H), 9.71 (s, 3H, 3N=CH), 12.04 (s, 3H, 3NH). UV λmax(ε): 310 (30831), 258 (47779), 222 (73777), 208 (58290) nm.


### 2.1.7. 1,3,5-Tri-{2-methoxy-4-[(3-phenyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4g)

Yield 1.06 g (98%). mp. 243°C. IR (KBr): 3210 (NH); 1750, 1716 (C=O); 1602, 1585 (C=N); 1268 (COO); 768 and 693 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.84 (s, 9H, 3OCH3), 7.50–7.64 (m, 18H, Ar-H), 7.90–7.94 (m, 6H, Ar-H), 9.02 (s, 3H, Ar-H), 9.72 (s, 3H, 3N=CH), 12.41 (s, 3H, 3NH). UV λmax(ε): 308 (24520), 258 (44728), 234 (67930), 216 (64853) nm.


## 2.1.1. 1,3,5-Tri-{2-methoxy-4-[(3-methyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4a)

Yield 0.67 g (74%). mp. 174°C. IR (KBr): 3223 (NH); 1751, 1715, 1705 (C=O); 1602 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 2.29 (s, 9H, 3CH3), 3.86 (s, 9H, 3OCH3), 7.45–7.68 (m, 9H, Ar-H), 9.02 (s, 3H, Ar-H), 9.75 (s, 3H, 3N=CH), 11.86 (s, 3H, 3NH). UV λmax(ε): 310 (41762), 262 (37725), 222 (64713) nm.


## 2.1.2. 1,3,5-Tri-{2-methoxy-4-[(3-ethyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4b)

Yield 0.72 g (76%). mp. 319°C. IR (KBr): 3220 (NH); 1750, 1703 (C=O); 1600 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 1.18 (t, 9H, 3CH3,J=7.52 Hz), 2.70 (q, 6H, 3CH2,J=7.52 Hz), 3.88 (s, 9H, 3OCH3), 7.34–7.69 (m, 9H, Ar-H), 9.02 (s, 3H, Ar-H), 9.75 (s, 3H, 3N=CH), 11.89 (s, 3H, 3NH). UV λmax(ε): 310 (22888), 260 (27033), 224 (55717) nm.


## 2.1.3. 1,3,5-Tri-{2-methoxy-4-[(3-n-propyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4c)

Yield 0.76 g (77%). mp. 168°C. IR (KBr): 3216 (NH); 1751, 1707 (C=O); 1598 (C=N); 1271 (COO)cm-1. 1H NMR (DMSO-d6): δ 0.94 (t, 9H, 3CH3,J=7.52 Hz), 1.68 (sext., 6H, 3CH2,J=7.52 Hz), 3.84 (s, 9H, 3OCH3), 2.64 (q, 6H, 3CH2,J=7.52 Hz), 7.41–7.66 (m, 9H, Ar-H), 9.00 (s, 3H, Ar-H), 9.74 (s, 3H, 3N=CH), 11,89 (s, 3H, 3NH). UV λmax(ε): 310 (23732), 260 (26373), 218 (52676) nm.


## 2.1.4. 1,3,5-Tri-{2-methoxy-4-[(3-benzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4d)

Yield 1.03 g (91%). mp. 161°C. IR (KBr): 3214 (NH); 1750, 1718 (C=O); 1599 (C=N); 1272 (COO); 760 and 706 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.85 (s, 9H, 3OCH3), 4.07 (s, 6H, 3CH2), 7.20–7.68 (m, 24H, Ar-H), 9.02 (s, 3H, Ar-H), 9.70 (s, 3H, 3N=CH), 12.03 (s, 3H, 3NH). UV λmax(ε): 310 (13919), 258 (17758), 216 (45657) nm.


## 2.1.5. 1,3,5-Tri-{2-methoxy-4-[(3-p-methylbenzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4e)

Yield 1.15 g (98%). mp. 216°C. IR (KBr): 3219 (NH); 1751, 1705 (C=O); 1600 (C=N); 1272 (COO); 829 (1,4-disubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 2.21 (s, 9H, 3CH3), 3.84 (s, 9H, 3OCH3), 4.00 (s, 6H, 3CH2), 7.06–7.65 (m, 21H, Ar-H), 9.02 (s, 3H, Ar-H), 9.69 (s, 3H, 3N=CH), 12.04 (s, 3H, 3NH). 13C NMR (DMSO-d6): δ 20.54 (3PhCH3), 30.83 (3CH2Ph), 56.07 (3OCH3), 111.93, 120.94 (arom-C), 123.50 (2C), 123.59 (2C), 128.57 (6C), 128.99 (6C), 130.35 (arom-C), 130.70 (3C), 132.68, 133.03 (arom-C), 135.46 (3C), 135.79 (3C), 141.13 (3C), 143.60 (3C), 146.36 (3C), 151.19 (3C), 150.95 (3triazole C3), 151.31 (N=CH), 151.91 (3triazole C5), 161.69 (3COO). UV λmax(ε): 300 (10113), 258 (13837), 226 (66208), 216 (61806) nm. Anal. Calcd. for C63H54N12O12 (1171.19): C, 64.61; H, 4.65; N, 14.35. Found: C, 64.85; H, 6.22; N, 13.29.


## 2.1.6. 1,3,5-Tri-{2-methoxy-4-[(3-p-chlorobenzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4f)

Yield 0.89 g (72%). mp. 230°C. IR (KBr): 3210 (NH); 1750, 1717 (C=O); 1598 (C=N); 1271 (COO); 824 (1,4-disubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.88 (s, 9H, 3OCH3), 4.09 (s, 6H, 3CH2), 7.35–7.56 (m, 24H, Ar-H), 9.02 (s, 3H, Ar-H), 9.71 (s, 3H, 3N=CH), 12.04 (s, 3H, 3NH). UV λmax(ε): 310 (30831), 258 (47779), 222 (73777), 208 (58290) nm.


## 2.1.7. 1,3,5-Tri-{2-methoxy-4-[(3-phenyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene  (4g)

Yield 1.06 g (98%). mp. 243°C. IR (KBr): 3210 (NH); 1750, 1716 (C=O); 1602, 1585 (C=N); 1268 (COO); 768 and 693 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 3.84 (s, 9H, 3OCH3), 7.50–7.64 (m, 18H, Ar-H), 7.90–7.94 (m, 6H, Ar-H), 9.02 (s, 3H, Ar-H), 9.72 (s, 3H, 3N=CH), 12.41 (s, 3H, 3NH). UV λmax(ε): 308 (24520), 258 (44728), 234 (67930), 216 (64853) nm.


## 2.2. General Procedure for the Synthesis of Compound5

The corresponding compound4 (0.001 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 40–45°C in vacuo and several recrystallizations of the residue from EtOH gave pure compounds 5 as colorless needles.


### 2.2.1. 1,3,5-Tri-{2-methoxy-4-[(1-acetyl-3-methyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene (5a)

Yield 0.66 g (64%). mp. 207°C. IR (KBr): 1728 (C=O); 1623, 1582 (C=N); 1268 (COO)cm-1. 1H NMR (DMSO-d6): δ 2.36 (s, 9H, 3CH3), 2.50 (s, 9H, 3COCH3), 3.85 (s, 9H, 3OCH3), 7.50–7.64 (m, 9H, Ar-H), 8.85 (s, 3H, Ar-H), 9.59 (s, 3H, 3N=CH). UV λmax(ε): 310 (20060), 258 (20237), 220 (63380), 216 (58300) nm.


### 2.2.2. 1,3,5-Tri-{2-methoxy-4-[(1-acetyl-3-benzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene (5d)

Yield 0.88 g (71%). mp. 182°C. IR (KBr): 1746 (C=O); 1602, 1590 (C=N); 1217 (COO); 746 and 710 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 2.50 (s, 9H, 3COCH3), 3.85 (s, 9H, 3OCH3), 4.16 (s, 6H, 3CH2), 7.23–7.58 (m, 24H, Ar-H), 9.01 (s, 3H, Ar-H), 9.58 (s, 3H, 3N=CH). UV λmax(ε): 310 (45812), 296 (45995), 258 (47147), 230 (83429), 224 (81387) nm.


## 2.2.1. 1,3,5-Tri-{2-methoxy-4-[(1-acetyl-3-methyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene (5a)

Yield 0.66 g (64%). mp. 207°C. IR (KBr): 1728 (C=O); 1623, 1582 (C=N); 1268 (COO)cm-1. 1H NMR (DMSO-d6): δ 2.36 (s, 9H, 3CH3), 2.50 (s, 9H, 3COCH3), 3.85 (s, 9H, 3OCH3), 7.50–7.64 (m, 9H, Ar-H), 8.85 (s, 3H, Ar-H), 9.59 (s, 3H, 3N=CH). UV λmax(ε): 310 (20060), 258 (20237), 220 (63380), 216 (58300) nm.


## 2.2.2. 1,3,5-Tri-{2-methoxy-4-[(1-acetyl-3-benzyl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-Benzene (5d)

Yield 0.88 g (71%). mp. 182°C. IR (KBr): 1746 (C=O); 1602, 1590 (C=N); 1217 (COO); 746 and 710 (monosubstituted benzenoid ring)cm-1. 1H NMR (DMSO-d6): δ 2.50 (s, 9H, 3COCH3), 3.85 (s, 9H, 3OCH3), 4.16 (s, 6H, 3CH2), 7.23–7.58 (m, 24H, Ar-H), 9.01 (s, 3H, Ar-H), 9.58 (s, 3H, 3N=CH). UV λmax(ε): 310 (45812), 296 (45995), 258 (47147), 230 (83429), 224 (81387) nm.


## 2.3. Antioxidant Activity: Chemicals

Butylated hydroxytoluene (BHT) was obtained from E. Merck (Merck KGaA, 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), and trichloroacetic acid (TCA) were obtained from Sigma (Sigma-Aldrich GmbH, Steinheim, Germany).


## 2.4. Reducing Power

The reducing power of the synthesized compounds was determined according to the method of Oyaizu [20]. 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%). 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 ×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.


## 2.5. Free Radical Scavenging Activity

Free radical scavenging activity of compounds was measured by DPPH•, using the method of Blois [21]. 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):
(1)Absorbance=0.0003×DPPH•-0.0174.
The capability to scavenge the DPPH radical was calculated using the following equation:
(2)DPPH•scavengingeffect(%)=(A0-A1A0)×100,
where A0 is the absorbance of the control reaction, and A1 is the absorbance in the presence of the samples or standards.


## 2.6. Metal Chelating Activity

The chelation of ferrous ions by the synthesized compounds and standards were estimated by the method of Dinis et al. [22]. 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 then the mixture was shaken vigorously and left standing at the 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+ 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.


## 2.7. Potentiometric Titrations

A Jenco model ion analyzer and an Ingold pH electrode were used for potentiometric titrations. For each compound that was 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 graph.


## 3. Results and Discussion

The 1,3,5-tri-{2-methoxy-4-[(3-alkyl/aryl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azo-methine]-phenoxycarbonyl}-benzene4a–gwere prepared. The starting compounds 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones 2a–gwere prepared from the reactions of the corresponding ester of ethoxycarbonylhydrazones 1a–gwith an aqueous solution of hydrazine hydrate as described in the literature [14, 19, 23]. Compounds 4were obtained from the reactions of compounds 3with 1,3,5-tri-(2-methoxy-4-formylphenoxycarbonyl)-benzene 1, which were synthesized by the reactions of 3-methoxy-4-hydroxybenzaldehyde with 1,3,5-benzenetricarbonyl chloride by using triethylamine. Then the reactions of compounds 4a and 4dwith acetic anhydride were investigated, and compounds 5a and 5dwere prepared (Scheme 1).Scheme 1The structures of seven new 1,3,5-tri-{2-methoxy-4-[(3-alkyl/aryl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-benzenes4a–g and two new 1,3,5-tri-{2-methoxy-4-[(1-acetyl-3-alkyl/aryl-4,5-dihydro-1H-1,2,4-triazol-5-on-4-yl)-azomethine]-phenoxycarbonyl}-benzenes 5a and 5d were characterized using IR, 1H NMR, 13C NMR, UV, and elemental analyses data.


### 3.1. Antioxidant Activity

The antioxidant activities of 9 new compounds4a–g,5a, and5dwere determined. Several methods have been used to determine antioxidant activities, and the methods used in the study are given below.


#### 3.1.1. Total Reductive Capability Using the Potassium Ferricyanide Reduction Method

The reductive capabilities of compounds were assessed by the extent of conversion of the Fe3+/ferricyanide complex to the Fe2+/ferrous form using the method of Oyaizu [20]. The reducing powers of the compounds were observed at different concentrations, and results were compared with BHA, BHT, and α-tocopherol. It has been observed that the reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [24]. 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 [25]. In this study, all the amount of the compounds showed lower absorbance than 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.


#### 3.1.2. DPPH Radical Scavenging Activity

The model of 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 [26]. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [27]. 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 resulted 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 [28]. In the study, antiradical activities of compounds and standard antioxidants such as BHA and α-tocopherol were determined by using DPPH• method. Scavenging effect values of compounds 4a, 4c, 4e, 4f, BHA and α-tocopherol at different concentrations are given in Figure 1. The newly synthesized compounds showed no activity as a radical scavenger.Figure 1
Scavenging effect of compounds4a, 4c, 4e, 4f, BHA, and α-tocopherol at different concentrations (12.5–25–37.5 μg/mL).


#### 3.1.3. Ferrous Ion Chelating Activity

The chelating effect towards ferrous ions by the compounds and standards was determined according to the method of Dinis [22]. 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 [29]. 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 [30] 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 [31]. Also, the production of highly active ROS such as O2•-, H2O2, and OH• is also catalyzed by free iron though Haber-Weiss reactions:
(3)O2•-+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 reactive free radicals via the Fenton reactions:(4)Fe2++H2O2⟶Fe3++OH-+OH•.Fe3+ ion also produces radicals from peroxides, even though the rate is tenfold less than that of Fe2+ ion, which is the most powerful pro-oxidant among the various types of metal ions [32]. Ferrous ion chelating activities of the compounds 4, 5, BHT, and BHA are shown in Figures 2 and 3, respectively. 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 ion [33]. The data obtained from Figures 2 and 3 reveal that the compounds, especially 4e, 5a, and 5ddemonstrate 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.Figure 2
Metal chelating effect of different amount of the compounds4a–g, BHT, and BHA on ferrous ions.Figure 3
Metal chelating effect of different amount of the compounds5a and 5d, BHT, and BHA on ferrous ions.


#### 3.1.4. Potentiometric Titrations

In order to determine thepKa values of the compounds 4a–g, they were titrated potentiometrically with TBAH in four non-aqueous solvents: isopropyl alcohol, tert-butyl alcohol, acetone, and DMF. The mV values read in each titration were plotted against 0.05 M TBAH volumes (mL) added, and potentiometric titration curves were obtained for all the cases. From the titration curves, the HNP values were measured, and the corresponding pKa 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 was studied [7, 8, 10, 12, 13, 17, 18].As an example for the potentiometric titration curves for 0.001 M solutions of compounds4b titrated with 0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, DMF and acetone are shown in Figure 4.Figure 4
Potentiometric titration curves of 0.001 M solutions of compound4b titrated with 0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone at 25°C.When the dielectric permittivity of solvents is taken into consideration, the acidity order can be given as follows: DMF(ε=36.7)> acetone (ε=36)> isopropyl alcohol (ε=19.4)>tert-butyl alcohol (ε=12). As seen in Table 1, the acidity order for compound 4ais: tert-butyl alcohol > acetone, for compound 4b it is  acetone > DMF > tert-butyl alcohol > isopropyl alcohol, for compounds 4cand4d it is  isopropyl alcohol > DMF, for compound 4e it is  tert-butyl alcohol > DMF > isopropyl alcohol, for compound 4f it is: isopropyl alcohol > DMF > acetone, while the order for compound 4 g is  acetone > DMF. Moreover, as seen in Table 1, for compounds 4a and 4 g in isopropyl alcohol, for compound 4ain DMF, for compounds 4c, 4d, 4f, and 4 g in tert-butyl alcohol, and for compounds 4c, 4d, and 4ein acetone, the HNP values and the corresponding pKa values were not obtained.Table 1
The HNP and the corresponding pKa values of compounds 4a-g in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone.
Comp.
Isopropyl alcohol
tert-Butyl alcohol
DMF
Acetone
HNP (mV)
pKa
HNP (mV)
pKa
HNP (mV)
pKa
HNP (mV)
pKa
4a
—
—
−142
10.00
—
—
−214
10.07
4b
−377
14.80
−343
14.17
−336
14.06
−146
9.81
4c
−172
9.73
—
—
−400
15.20
—
—
4d
−253
11.47
—
—
−364
14.67
—
—
4e
−432
16.08
−195
10.45
−398
15.34
—
—
4f
−244
11.75
—
—
−467
15.66
−609
—
4g
—
—
—
—
−730
—
−160
9.87As 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 [7, 8, 10, 12, 13, 17, 18, 34]. Table 1 and Figure 4 show that the HNP values and corresponding pKa 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.


## 3.1. Antioxidant Activity

The antioxidant activities of 9 new compounds4a–g,5a, and5dwere determined. Several methods have been used to determine antioxidant activities, and the methods used in the study are given below.


### 3.1.1. Total Reductive Capability Using the Potassium Ferricyanide Reduction Method

The reductive capabilities of compounds were assessed by the extent of conversion of the Fe3+/ferricyanide complex to the Fe2+/ferrous form using the method of Oyaizu [20]. The reducing powers of the compounds were observed at different concentrations, and results were compared with BHA, BHT, and α-tocopherol. It has been observed that the reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [24]. 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 [25]. In this study, all the amount of the compounds showed lower absorbance than 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.


### 3.1.2. DPPH Radical Scavenging Activity

The model of 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 [26]. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [27]. 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 resulted 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 [28]. In the study, antiradical activities of compounds and standard antioxidants such as BHA and α-tocopherol were determined by using DPPH• method. Scavenging effect values of compounds 4a, 4c, 4e, 4f, BHA and α-tocopherol at different concentrations are given in Figure 1. The newly synthesized compounds showed no activity as a radical scavenger.Figure 1
Scavenging effect of compounds4a, 4c, 4e, 4f, BHA, and α-tocopherol at different concentrations (12.5–25–37.5 μg/mL).


### 3.1.3. Ferrous Ion Chelating Activity

The chelating effect towards ferrous ions by the compounds and standards was determined according to the method of Dinis [22]. 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 [29]. 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 [30] 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 [31]. Also, the production of highly active ROS such as O2•-, H2O2, and OH• is also catalyzed by free iron though Haber-Weiss reactions:
(3)O2•-+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 reactive free radicals via the Fenton reactions:(4)Fe2++H2O2⟶Fe3++OH-+OH•.Fe3+ ion also produces radicals from peroxides, even though the rate is tenfold less than that of Fe2+ ion, which is the most powerful pro-oxidant among the various types of metal ions [32]. Ferrous ion chelating activities of the compounds 4, 5, BHT, and BHA are shown in Figures 2 and 3, respectively. 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 ion [33]. The data obtained from Figures 2 and 3 reveal that the compounds, especially 4e, 5a, and 5ddemonstrate 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.Figure 2
Metal chelating effect of different amount of the compounds4a–g, BHT, and BHA on ferrous ions.Figure 3
Metal chelating effect of different amount of the compounds5a and 5d, BHT, and BHA on ferrous ions.


### 3.1.4. Potentiometric Titrations

In order to determine thepKa values of the compounds 4a–g, they were titrated potentiometrically with TBAH in four non-aqueous solvents: isopropyl alcohol, tert-butyl alcohol, acetone, and DMF. The mV values read in each titration were plotted against 0.05 M TBAH volumes (mL) added, and potentiometric titration curves were obtained for all the cases. From the titration curves, the HNP values were measured, and the corresponding pKa 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 was studied [7, 8, 10, 12, 13, 17, 18].As an example for the potentiometric titration curves for 0.001 M solutions of compounds4b titrated with 0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, DMF and acetone are shown in Figure 4.Figure 4
Potentiometric titration curves of 0.001 M solutions of compound4b titrated with 0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone at 25°C.When the dielectric permittivity of solvents is taken into consideration, the acidity order can be given as follows: DMF(ε=36.7)> acetone (ε=36)> isopropyl alcohol (ε=19.4)>tert-butyl alcohol (ε=12). As seen in Table 1, the acidity order for compound 4ais: tert-butyl alcohol > acetone, for compound 4b it is  acetone > DMF > tert-butyl alcohol > isopropyl alcohol, for compounds 4cand4d it is  isopropyl alcohol > DMF, for compound 4e it is  tert-butyl alcohol > DMF > isopropyl alcohol, for compound 4f it is: isopropyl alcohol > DMF > acetone, while the order for compound 4 g is  acetone > DMF. Moreover, as seen in Table 1, for compounds 4a and 4 g in isopropyl alcohol, for compound 4ain DMF, for compounds 4c, 4d, 4f, and 4 g in tert-butyl alcohol, and for compounds 4c, 4d, and 4ein acetone, the HNP values and the corresponding pKa values were not obtained.Table 1
The HNP and the corresponding pKa values of compounds 4a-g in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone.
Comp.
Isopropyl alcohol
tert-Butyl alcohol
DMF
Acetone
HNP (mV)
pKa
HNP (mV)
pKa
HNP (mV)
pKa
HNP (mV)
pKa
4a
—
—
−142
10.00
—
—
−214
10.07
4b
−377
14.80
−343
14.17
−336
14.06
−146
9.81
4c
−172
9.73
—
—
−400
15.20
—
—
4d
−253
11.47
—
—
−364
14.67
—
—
4e
−432
16.08
−195
10.45
−398
15.34
—
—
4f
−244
11.75
—
—
−467
15.66
−609
—
4g
—
—
—
—
−730
—
−160
9.87As 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 [7, 8, 10, 12, 13, 17, 18, 34]. Table 1 and Figure 4 show that the HNP values and corresponding pKa 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.


## 3.1.1. Total Reductive Capability Using the Potassium Ferricyanide Reduction Method

The reductive capabilities of compounds were assessed by the extent of conversion of the Fe3+/ferricyanide complex to the Fe2+/ferrous form using the method of Oyaizu [20]. The reducing powers of the compounds were observed at different concentrations, and results were compared with BHA, BHT, and α-tocopherol. It has been observed that the reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [24]. 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 [25]. In this study, all the amount of the compounds showed lower absorbance than 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.


## 3.1.2. DPPH Radical Scavenging Activity

The model of 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 [26]. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [27]. 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 resulted 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 [28]. In the study, antiradical activities of compounds and standard antioxidants such as BHA and α-tocopherol were determined by using DPPH• method. Scavenging effect values of compounds 4a, 4c, 4e, 4f, BHA and α-tocopherol at different concentrations are given in Figure 1. The newly synthesized compounds showed no activity as a radical scavenger.Figure 1
Scavenging effect of compounds4a, 4c, 4e, 4f, BHA, and α-tocopherol at different concentrations (12.5–25–37.5 μg/mL).


## 3.1.3. Ferrous Ion Chelating Activity

The chelating effect towards ferrous ions by the compounds and standards was determined according to the method of Dinis [22]. 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 [29]. 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 [30] 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 [31]. Also, the production of highly active ROS such as O2•-, H2O2, and OH• is also catalyzed by free iron though Haber-Weiss reactions:
(3)O2•-+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 reactive free radicals via the Fenton reactions:(4)Fe2++H2O2⟶Fe3++OH-+OH•.Fe3+ ion also produces radicals from peroxides, even though the rate is tenfold less than that of Fe2+ ion, which is the most powerful pro-oxidant among the various types of metal ions [32]. Ferrous ion chelating activities of the compounds 4, 5, BHT, and BHA are shown in Figures 2 and 3, respectively. 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 ion [33]. The data obtained from Figures 2 and 3 reveal that the compounds, especially 4e, 5a, and 5ddemonstrate 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.Figure 2
Metal chelating effect of different amount of the compounds4a–g, BHT, and BHA on ferrous ions.Figure 3
Metal chelating effect of different amount of the compounds5a and 5d, BHT, and BHA on ferrous ions.


## 3.1.4. Potentiometric Titrations

In order to determine thepKa values of the compounds 4a–g, they were titrated potentiometrically with TBAH in four non-aqueous solvents: isopropyl alcohol, tert-butyl alcohol, acetone, and DMF. The mV values read in each titration were plotted against 0.05 M TBAH volumes (mL) added, and potentiometric titration curves were obtained for all the cases. From the titration curves, the HNP values were measured, and the corresponding pKa 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 was studied [7, 8, 10, 12, 13, 17, 18].As an example for the potentiometric titration curves for 0.001 M solutions of compounds4b titrated with 0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, DMF and acetone are shown in Figure 4.Figure 4
Potentiometric titration curves of 0.001 M solutions of compound4b titrated with 0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone at 25°C.When the dielectric permittivity of solvents is taken into consideration, the acidity order can be given as follows: DMF(ε=36.7)> acetone (ε=36)> isopropyl alcohol (ε=19.4)>tert-butyl alcohol (ε=12). As seen in Table 1, the acidity order for compound 4ais: tert-butyl alcohol > acetone, for compound 4b it is  acetone > DMF > tert-butyl alcohol > isopropyl alcohol, for compounds 4cand4d it is  isopropyl alcohol > DMF, for compound 4e it is  tert-butyl alcohol > DMF > isopropyl alcohol, for compound 4f it is: isopropyl alcohol > DMF > acetone, while the order for compound 4 g is  acetone > DMF. Moreover, as seen in Table 1, for compounds 4a and 4 g in isopropyl alcohol, for compound 4ain DMF, for compounds 4c, 4d, 4f, and 4 g in tert-butyl alcohol, and for compounds 4c, 4d, and 4ein acetone, the HNP values and the corresponding pKa values were not obtained.Table 1
The HNP and the corresponding pKa values of compounds 4a-g in isopropyl alcohol, tert-butyl alcohol, DMF, and acetone.
Comp.
Isopropyl alcohol
tert-Butyl alcohol
DMF
Acetone
HNP (mV)
pKa
HNP (mV)
pKa
HNP (mV)
pKa
HNP (mV)
pKa
4a
—
—
−142
10.00
—
—
−214
10.07
4b
−377
14.80
−343
14.17
−336
14.06
−146
9.81
4c
−172
9.73
—
—
−400
15.20
—
—
4d
−253
11.47
—
—
−364
14.67
—
—
4e
−432
16.08
−195
10.45
−398
15.34
—
—
4f
−244
11.75
—
—
−467
15.66
−609
—
4g
—
—
—
—
−730
—
−160
9.87As 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 [7, 8, 10, 12, 13, 17, 18, 34]. Table 1 and Figure 4 show that the HNP values and corresponding pKa 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.


## 4. Conclusion

The synthesis andin 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. The data reported with regard to the observed radical scavenging and metal chelating activities of the studied compounds 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. These results may also provide some guidance for the development of novel triazole-based therapeutic target.



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*Source: 517420-2012-08-14.xml*