Synthesis and Antioxidant Ability of New 5-amino-1,2,4-Triazole Derivatives Containing 2,6-dimethoxyphenol

Introduction  

The reactive oxygen species (ROS) and other related free radicals species are witty to react either directly or indirectly, to damage all biomolecules. This damage can cause many diseases ¹, such as inflammatory², cancers³, degenerative⁴ and chronic diseases⁵. The antioxidant compounds are the most important species which can inhibit the oxidative stress in biological system and prevent any free radicals damage. The phenolic compounds  are one of the significant antioxidant type, as well reported it possess  broad biological activity such as  anti inflammatory⁶ , anticancer⁷  and gastroprotective⁸.

Generally, antioxidants compounds donate protons to become stable free radicals. This stability increases with the extent of delocalization and enhances the antioxidant ability^{9, 10} . As such, many synthesized compounds containing long chain resonance exhibited significant antioxidant activity. Furthermore, the compounds which can be considered a strong antioxidant usually possess common structural features. They often own multiple phenolic hydroxyl groups like flavonoids ¹¹ or which have full conjugation π system like carotenoids¹². Moreover, exhibited substituted groups might influence on the scavenging ability. This indicates the existence of a close relationship between the chemical structure and the ability to scavenge free radicals.

Derivatives of 1,2,4-triazole exhibited various types of biological activities e.g. Antimicrobial¹³, antiviral,^{14, 15} inhibitors of Methionine Aminopeptidase-2,¹⁶ Anhydrase inhibitors,¹⁷ anti-cancer¹⁸, anti-inflammatory,¹⁹ inhibitors of the HIV-1,²⁰ analgesic ²¹ and antioxidant ²².As well the compounds of Schiff base derivatives displayed broader properties and applications such as nonlinear optical devices ²³ liquid crystalline ^{24, 25},  dyes ²⁶, biological ^27-29 and pharmaceutical ³⁰ applications.

The 2,6-dimethoxyphenol derivatives won wide attention in recent years as antioxidants^{31, 32} . In this work we presented the synthesis of new  4-((arylidine)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)phenyl)-1,2,4-triazole-5-thione (7a-7f) as new antioxidant material.

Material and Method

Chemistry

The IR spectra were obtained with a Perkin Elmer 400 Fourier Transform Infrared (FTIR) Spectrometer. ¹H and ¹³C-NMR spectra were recorded at Joel Lambda spectrometers at 400 MHz)  UM, Malaysia). DMSO-d₆ were used as solvents with TMS as the internal standard. The mass spectra were recorded using an Agilent 5975 system for EI/MS and a Finnigan TSQ7000 for HREIMs (NUS, Singapore). For UV spectroscopy, a Power Wave X340 (BIO-TEK Instruments, Inc., Winooski, VT, USA) was used to record the FRAP and DPPH.  Melting points were measured on a Sturt-SMP10 melting point apparatus in open-end capillary tubes. Flash column chromatography on silica gel 60 (230–400 mesh, E. Merck) was employed. General grade solvents and reagents were purchased from commercial suppliers and used without further purification.

4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzoic acid  4

This compound was synthesized according to the procedure described by K. F. Ali ³³.Recrystallized with  ethanol to obtain white microcrystals. Yield 60 %, Mp 150-152  ̊C [ lit. 152-154 ̊C ³³], IR (KBr) v_max 3354 (OH), 3046 (CH_Ar), 2972, 2893 (CH_aliphatic), 1676 (C=O) 1585 (C=C), 1180 (Ar-O-C), cm^-1.  ¹H-NMR (400MHz, CDCl₃): 3.87(6H,  s, 2×OCH₃), 4.33 (2H, s, OCH₂), 4.38 (2H, s, OCH₂),  6.66 (s, 2H, H-3), 7.65 ( 2H, d, J 8.2, H-8),8.12( 2H, d, J 8.0, H-9). 9.15 (1H, bs, OH), ¹³C-NMR (100 MHz, CDCl₃): 56.11(2C, OCH₃), 71.22(1C, CH₂OCH₂), 72.77 (1C, CH₂OCH₂), 109.05(2C, CH), 128.84(2C,CH), 129.37(1C),  131.21(2C, CH), 134.23 (1C), 139.65 (1C), 142.13(1C) 152.01 (2C), 169.58 (1C, C=O) HREIMs m/z =  318.1100 [M˙⁺] (calc. for C₁₇H₁₈O₆, 318.1103).

4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzohydrazide 5

This compound was synthesized according to the procedure described by K. F. Ali ³³.The crude was recrystallized from ethanol to give white solid. Yield 87%, Mp 94-96 ̊ C [ lit. 92-94 ̊C ³³] , IR (KBr) v_max 3422 (OH _phenol), 3308, 3218 (NH, NH₂), 3037 (CHAr), 2975 -2885 (CHaliphatic), 1661 (C=O), 1585 (C=C), 1151(Ar-O-C), cm^-1. ¹H-NMR (400MHz, DMSO-d₆):  3.71 (6H,  s, 2× OCH₃), 3.93 (2H, s, OCH₂), 4.09 (2H, s, OCH₂), 4.63 (bs, 2H, NH₂)  6.52 (s, 2H, H-3), 7.67   ( 2H, d, J 8.1, H-8), 8.22( 2H, d, J 7.92, H-9), 8.863 (1H, bs, CONH), 9.21(1H, bs, OH), ¹³C-NMR (100 MHz, DMSO-d₆): 56.3 (2C, OCH₃), 71.14 (1C, CH₂OCH₂), 72.60 (1C, CH₂OCH₂), 106.88(2C, CH), 129.11(2C,CH), 128.92(1C),  130.01(2C, CH), 133.42(1C), 139.04(1C), 141.55(1C) 152.01 (2C), 166.15(C=O). HREIMs m/z = 332.1369 [M˙⁺] (calc. for C₁₇H₂₀N₂O₅, 332.1372)

General synthesis of 4-amino-3-(4-(((4-hydroxy-3,5dimethoxybenzyl)oxy)methyl)phenyl) -1,2,4-triazole-5-thione. 6

Method A

4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzoic acid (3.2gm 10.05,mmol) and Thiocar-bonyldihydrazide (1.17 gm , 11 mmol) was mixed then grinded after that the mixture was transferred to conical flask and heated until melt at 160-165 ˚C for three hour. The mixture left to cool to ambient temperature.The crude was  boiled with (2×20 mL) dichloromethane then filtrated. The organic combined evaporated under reduced pressure. The crude precipitated was recrystallized from ethanol to afford white crystals. Yield 51% Mp  68-70 ˚C,

Method B

To a solution of 4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzohydrazide (3.33 g, 10 mmol) in xx mL absolute ethanol was added. Carbon disulphide (0.92 g, 12 mmol) was added and potassium hydroxide (0.56 g, 10 mmol) at room temperature. The mixture was stirred for 24 h, then 25 mL dry ether was added and allow to stir for another 2 h. The precipitated was filtrated and washed with dry ether. The product was dried at 80 °C to give white solid of potassium 2-(4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzoyl)hydrazinecarbodithioate salt (4 g,9 mmol). The resulting product was dissolved in excess of hydrazine hydrate 80%. The mixture was heated and refluxed for 7 h then cooled and poured into ice water. The pH was adjusted to 5 by using 10% HCl. The precipitate was filtrated, washed with water, dried and recrystallized from ethanol to obtain yield 68% of white crystals. Mp  68-70.

IR (KBr) v_max 3507 (OH _phenol), 3410, 3330and  3170 (NH, NH₂), 3013 (CHAr), 2995 -2887 (CHaliphatic), 1628 (C=N), 1585 (C=C), 1362(C-N), 1245(C=S), 1201(Ar-O-C), cm^-1. ¹H-NMR (400MHz, DMSO-d₆): 3.82 (6H,  s, 2× OCH₃), 3.89 (2H, s, OCH₂), 3.98 (2H, s, OCH₂), 6.13 (bs, 2H, NH₂)  6.58 (s, 2H, H-3), 7.69 (2H, d, J 8.0, H-8), 8.25( 2H, d, J 7.82, H-9), 9.27(1H, bs, OH),14.03(1H, bs, NH). ¹³C-NMR (100 MHz, DMSO-d₆): 57.21, (2C, OCH₃), 72.04 (1C, CH₂OCH₂), 72.69 (1C, CH₂OCH₂), 107.17(2C, CH), 128.78(2C,CH), 129.90(1C),  131.10 (2C, CH), 133.72(1C), 138.86 (1C), 142.53(1C) 149.54 (1C,C=N), 152.01 (2C), 166.61(C=S). HREIMs m/z = 388.1202 [M˙⁺] (calc. for C18H20N4O4S, 388.1205)

General synthesis of 4-((arylidine)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)phenyl)-1,2,4-triazole-5-thione 7a-7f

aryl aldehyde (0.51mmol) was added to a stirred solution of 4-amino-3-(4-(((4-hydroxy-3,5dimethoxybenzyl)oxy)methyl)phenyl) -1,2,4-triazole-5-thione (0.2 gm, 0.51mmol) in 5 mL glacial acetic acid and the mixture was refluxed for 9h. After cooling the mixture was poured into 50 mL crushed ice. the precipitate collected, washed with cold distilled water and recrystallized from suitable solvent.

4-((4-hydroxybenzylidene)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy) methyl)phenyl)-1,2,4-triazole-5-thione 7a

Recrystallized from methanol to give pale yellow precipitate.Yield 83%, Mp 115-117 ˚C  IR (KBr) v_max, 3498(OH _phenol), 3152(NH), 3062(CH_Aromatic), 1623 (CH=N), 1607 (C=N_triazole), 1995, 1985(C=C), 1247(C=S), 1201(C-O), cm-¹, ¹H-NMR (DMSO-d₆, δ): 3.62(6H,  s, 2× OCH₃), 4.34 (2H, s, OCH₂), 4.52 (2H, s, OCH₂),  6.67 (s, 2H, H-3), 6.84( 2H, d,  J 8.2), 7.86 (4H, d, J 8.4), 7.93 ( 2H, d, J 8.2), 8.24( 2H, d, J 8.21,), 8.46(1H, s ,CH=N), 9.22(1H,bs, OH), 9.95 (bs, 2H, OH),14.21(1H, bs, NH) ¹³C-NMR (100 MHz, DMSO-d₆): 56.09(2C, OCH₃), 70.88(1C, CH₂OCH₂), 69.90(1C, CH₂OCH₂), 107.85(2C, CH), 116.11(2C), 125.56(1C) 129.07(2C, CH), 129.25(1C), 130.47(2C), 131.23(2C, CH), 133.09 (1C), 138.85(1C), 142.17 (1C) 151.91 (2C), 157(1C), 160.31(CH=N), 160.93 (C=N _triazole),182.43(1C, C=S). HREIMs m/z =492.1461 [M˙⁺] (calc. for C₂₅H₂₄N₄O₅S, 492.1467).

4-((4-hydroxy-3-methoxybenzylidene)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)phenyl) -1,2,4-triazole-5-thione 7b

Recrystallized from ethanol to afford yellow precipitate. Yield 78% Mp 81-83 ˚C IR (KBr) v_max, 3568(OH _phenol), 3169(NH), 3053(CH_Ar), 1621 (CH=N), 1605 (C=N_triazole), 1595, 1983(C=C), 1232(C=S), 1195(C-O); cm-¹. ¹H NMR (DMSO-d₆, δ): 3.57(6H,  s, 2× OCH₃), 3.89 (s, 3H, OCH₃),  4.41 (2H, s, OCH₂), 4.59 (2H, s, OCH₂),  6.62 (s, 2H, H-3), 6.83 (d, 1H, J 7.79),  6.86( 2H, d, J 8.0), 7.26 (dd, 2H, J 8.22;1.8), 7.43 (s, 1H)) 7.91 ( 2H, d, J 8.22), 8.22( 2H, d, J 8.2), 8.59 (s, 1H, CH=N), 9.23(1H, bs, OH), 10.06 (1H bs, , OH),14.01(1H, bs, NH). ¹³C-NMR (100 MHz, DMSO-d₆): 56.09(2C, OCH₃), 56.13(1C,OCH₃), 70.91(1C, CH₂OCH₂), 69.78(1C, CH₂OCH₂), 108.04(2C, CH), 110.31(1C), 116.1(1C)  116.37(2C), 124.25 (1C), 125.63(1C), 126.08(1C), 129.11(2C, CH), 129.19(1C), 130.52(2C), 131.53(2C, CH), 133.29(1C), 138.65(1C), 142.17 (1C), 148.45(1C), 151.92 (2C), 153.39(1C), 157.1(1C), 161.10(CH=N), 164.22(C=N _triazole), 179.93(1C, C=S). HREIMs m/z = 522.1568 [M˙⁺] (calc. for C₂₆H₂₆N₄O₆S, 522.1573).

4-((4-hydroxy-3-ethoxybenzylidene)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)phenyl) -1,2,4-triazole-5-thione 7c

The crude product was recrystallized from acetonitrile to afford yellow crystals. Yield 71% Mp 104-106 ˚C. . IR (KBr) v_max, 3584(OH _phenol), 3170(NH), 3037(CH_Ar), 2987, 2865 (CH _aliphatic)  1618 (CH=N), 1607 (C=N_triazole), 1595, 1983(C=C), 1238(C=S). 1201(C-O), cm-¹. ¹H-NMR (400MHz, DMSO-d₆)  )  1.43 (t, 3H, J 6.9, O-CH₂CH₃), 3.72(6H,  s, 2× OCH₃), 3.92 (q, 2H, J 7.1, O-CH₂), 4.39 (2H, s, OCH₂), 4.74 (2H, s, OCH₂),  6.54 (s, 2H, H-3), ), 6.85 (d, 2H, J 8.0, 7.24 (1H, dd, J 8.1; 1.68) 7.71   ( 2H, d, J 8.22, H-8), 8.26( 2H, d, J 8.2,), 8.59 (1H, s, CH=N), 9.16(1H, bs, OH), ), 9.78 (1H, bs, OH),14.14(1H, bs, NH), ¹³C-NMR (100 MHz, DMSO-d₆): 14.94 (1C, O-CH₂CH_3), 55.21(2C, OCH₃), 64.32 (1C, OCH₂), 70.96(1C, CH₂OCH₂), 69.87(1C, CH₂OCH₂), 108.05(2C, CH), 111.32(1C), 117.13 (1C), 124.77 (1C), 127.51(1C), 129.39(2C, CH), 129.65(1C), 132.36(2C, CH), 134.16(1C), 138.82(1C), 142.72 (1C), 147.86 1C), 150.61 (1C) 151.89 (2C), 161.01(1C, CH=N), 163.12 (C=N _triazole), 181.34(1C, C=S). HREIMs m/z =  5536.1725 [M˙⁺] (calc. for C₂₇H₂₈N₄O₆S, 536.1730).

4-((4-hydroxy-3,5-dimethoxybenzylidene)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)phenyl) -1,2,4-triazole-5-thione 7d

Crude product recrystallized from chloroform-ethanol to afford pale yellow precipitate. Yield 73%. Mp 136-138 °C. IR (KBr) v_max ,3605(OH_phenol), 3172(NH), 3062(CH_Ar), 2984, 2890 (CH _aliphatic) 1614 (CH=N), 1605 (C=N_triazole), 1595, 1980(C=C), 1221(C=S), 1198(C-O), , cm^-1, ¹H-NMR (400MHz, DMSO-d₆)3.73(6H,  s, 2× OCH₃), 3.85 (s, 6H, 2×OCH₃) 4.42 (2H, s, OCH₂), 4.56 (2H, s, OCH₂),  6.59 (s, 2H, H-3), 7.24 (s, 2H) 7.71 ( 2H, d, J 8.2, H-8), 8.28( 2H, d, J 8.0,), 8,51(1H, s, CH=N), 9.08(2H, bs, OH),9.65(1H, bs, OH),13.97(1H, bs, NH)¹³C-NMR (100 MHz, DMSO-d₆): 56.28(2C, OCH₃), 56.31(2C, OCH₃) 70.17(1C, CH₂OCH₂), 68.99(1C, CH₂OCH₂), 106.07 (2C,  CH), 107.96(2C, CH), 129.27(2C, CH), 124.78(1C) 129.28(1C),  131.23(2C, CH), 133.43(1C), 138.66(1C), 142.41 (2C) 151.85 (4C), 160.35(1C, CH=N), 162.13 (1C, C=N_triazole),181.22(1C, C=S). HREIMs m/z = 552.1673 [M˙⁺] (calc. for  C₂₇H₂₈N₄O₇S, 552.1679).

4-((4-hydroxy-3,5-di-tert-butylbenzylidene)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)phenyl) -1,2,4-triazole-5-thione 7e

The crude products recrystallized from Chloroform afforded off white precipitate. Yield 60%, mp 158-160, IR (KBr) v_max, 3599.7 (OH_phenol), 3177(NH), 3068 (CH_Ar), 2990-2885 (CH_alph), 1622(CH=N), 1608(C=N_triazole), 1595-1589 (C=C) and 1262(C=S). ¹H NMR, δ 3333334.65 (2H, s, OCH₂), 5.62 (1H, bs, OH), 6.64 (2H, s, H-3), 7.62  ( 2H, d, J 8.22, H-8), 7.91(s, 2H),  8.18( 2H, d, J 8.1,), 8.60(1H,s, CH=N), 9.14(1H, bs, OH),(1H, bs, NH) ¹³C-NMR (100 MHz, DMSO-d₆): 30.37(6C, 4(C(CH₃)₃), 34.46(2C, C(CH₃)₃)) 56.11(2C, OCH₃), 71.24(1C, CH₂OCH₂), 69.93(1C, CH₂OCH₂), 107.65(2C, CH), 116.86 (1C) , 124.80 (1C), 129.07(2C, CH), 129.35(1C),  131.38(2C, CH), 133.14(1C), 138.85(1C), 142.17 (1C) 151.79 (2C), 157.15 (1C), 161.77 (1C, CH=N), 165.11 (C=N _triazole),182.71(1C,C=S). HREIMs m/z = 604.2715 [M˙⁺] (calc. for C₃₃H₄₀N₄O₅S, 604.2719).

4-((2-hydroxy-3,5-di-tert-butylbenzylidene)amino)-3-(4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)phenyl) -1,2,4-triazole-5-thione 7f

The crude products recrystallized from Chloroform-ethanol afforded pale yellow precipitate. Yield 73 %, mp 122-124˚C, IR (KBr) v_max, 3603 (OH_phenol), 3173(NH),  3068 (CH_Ar), 2980-2885 (CH_alph), 1621(CH=N), 1604(C=N_triazole), 1597-1589 (C=C) and 1262(C=S) ¹H NMR, δ (ppm, CDCl₃, 400 MHz): )1.32(9H, s, o-(t-Bu),1,45(9H, s, p-(t-Bu), 3.71(6H, s, 2× OCH₃), 4.42 (2H, s, OCH₂), 4.45 (2H, s, OCH₂), 6.57 (s, 2H, H-3),7.47(1H,d, J 2.4) 7.52(1H, d, J 2.2)7.74  ( 2H, d, J 8.22, H-8), 8.18( 2H, d, J 8.2,),8,56(1H, s, CH=N) 9.14(1H,bs, OH),10.21 1H,bs,OH), 14.29(1H,bs,NH), ¹³C-NMR (100 MHz, DMSO-d₆): 29.32(3C, p-(C(CH₃)₃), 31.52(3C, o-(C(CH₃)₃), 34.56(1C,o– C(CH₃)₃),  35.41(1C,p– C(CH₃)₃56.31(2C, OCH₃), 71.02 (1C, CH₂OCH₂), 72.57 (1C, CH₂OCH₂), 106.35(1C), 108.95(2C, CH), 120.41 (1C),128.29(1C), 128.81(2C,CH), 129.32(1C),  131.24(2C, CH), 134.13 (1C),137.61(1C), 139.55 (1C), 141.7(1C), 142.17(1C) 151.91 (2C),154(1C), 162.07 (1C, CH=N), 164.08 (C=N _triazole),182.88(1C,C=S) HREIMs m/z = 604.2716 [M˙⁺] (calc. for C₃₃H₄₀N₄O_sS, 604.2719)

Antioxidant

DPPH assay

The assay was performed as reported by Gerhauser et al. ³⁴. Five microliters of the sample (dissolved in ethanol) was added into 195 μL of 100 μM DPPH reagent in ethanol (96%) and mixed in a 96-well plate. The intensity of the colour was measured for 3 h at an interval of 20 min at 515 nm. Ascorbic acid and BHT were used as reference.

FRAP assay

The FRAP assay was performed according to the Benzie and Strain ³⁵ method. The FRAP reagent was prepared by combining 300 mM acetate buffer and 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl and 20 mM FeCl3·6H2O, in a ratio of 10:1:1. The FRAP reagent wasincubated at 37 °C prior to use. Ten microliters of the sample was reconstituted in the carrier (solventor ultrapure water) and mixed with 300 μL of FRAP reagent. The mixture was incubated at 37 °C for 4 min in a microplate reader. The absorbance of the complex was 593 nm. The FRAP value can be calculated using the following equation³⁶ :

FRAP = [(0–4 min ∆A593 nm of test sample)/(0–4 min ∆A593 nm of standard)]

× [standard] (µM) × Y × 1000

Where; Y is absorbance of the spectrophotometer.

Results and Discussion 

Chemistry

The 4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzoic acid 4 and their corresponded acid hydrazide 5 were synthesized from according to the procedure described by K. F. Ali ³³ as depicted in Scheme 1

Scheme 1: Synthetic route of4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzoic acid 4 and theircorresponded acid hydrazide.5   Click here to View scheme

 

The 4-amino-3-(4-(((4-hydroxy-3,5dimethoxybenzyl)oxy)methyl)phenyl)-1,2,4-triazole-5-thione. 6 were synthesized from two different methods. The first one, from melt reaction with Thiocarbonyldihydrazide for tree hour at 160-165 ˚C to obtained 51% yield. The second methods, was from converted the corresponded acid hydrazide 5  to their corresponded potassium hydrazinecarbodithioate salt as the first step. The next step was reacted  this salt with hydrazine hydrate under reflux for 7h. this method exhibited yield higher than method one. The compound 7a-7f were synthesized from reaction compound 6 with hydroxyl substituted benzaldehyde in the presence of glacial acetic acid. As depicted in Scheme 2

Scheme 2: Synthetic route of compound 6 and 7a-7f   Click here to View scheme

 

All synthesized compounds were characterized by IR, ¹H-NMR, ¹³C-NMR spectrum beside the EIMS and HRMIS. The IR spectrum of compound 4 displayed rise a new band of C=O at 1676 cm^-1 and disappeared the band of C≡N at 2243 cm^-1.compound 5 showed shifting at carbonyl group which is appeared at 1661 cm^-1 of acid to hydrazide as well new bands of NH and NH₂ were appeared at 3308-3218 cm^-1. The IR spectrum of compound 6 exhibited disappeared of carbonyl group and new interesting bands were appeared at 1628 cm^-1 corresponded the C=N group of the triazole ring as and at 1245 cm^-1 of C=S beside the value of NH and NH2 bands were shifted to 3410,3330 and 3170 cm^-1 respectively. The spectra of 7a-7f displayed disappeared the band of NH2 and a new interesting band for the Schiff base was appeared at 1623-1614 cm^-1  besides the C=N of the triazole ring which appeared at 1608-1604 cm^-1. The ¹H-NMR spectrum of compound 4 showed disappearing of tri methylsilyl group at 0.17 and appears of the broad singlet of OH for the converting the nitrile group to carboxylic acid. The ¹H-NMR spectrum of compound 5 showed the disappearing hydroxyl group of the carboxylic as well a new two board singlet of NH2 and NH were appeared at 4.63 δ and 8.86 δ respectively. The ¹H-NMR of compound 6 displayed all expected peaks in triazole structure. The ¹H-NMR spectra of compounds 7a-7f showed the appearance of new peaks represented the Schiff base (imin group) and the expected proton of the substituted hydroxybenzylidene (see the experimental suction) also appeared in their expected rang. The ¹³C-NMR spectrum of compound 4 exhibited two carbons of CH₂OCH₂ at 3.87 & 433 δ, beside disappearing the carbon of C≡N and raised a new carbon of C=O. That enhances the evidence of successful convert compound 3 to 4 besides the spectra of  IR and ¹H-NMR. The 13C-NMR showed change in value of carbonyl of compound 5 due to convert the acid to acid hydrazide. The spectrum of compound 6 showed two new carbons first on the C=N at 149.54 δ of triazole ring and the second one at 166.61 δ represented the C=S. The ¹³C spectra of  compounds 7a-7f were showed an interested peak of CH=N beside the carbons of the hydroxybenzylidene. The substituent group for 7b-7f were appeared in the expected range. The EIMS spectra showed the molecular ion M^•+ for all compounds and the base peak (100%) as well the HREIMs value was confirmed the accurate mass and the molecular formula .as depicted in Table 1.

Table 1: The hydroxyl substituted and selected properties of synthesized compound 7a-7f  Click here to View table

 

Antioxidant Activity

The synthesized compound 6 and7a-7f were showed  high antioxidant ability in both assays(DPPH and FRAP). Compound 6 showed antioxidant ability higher than 7a-7b in both assays and that could attributed to exhibited a pro-oxidative effect³⁷. Even though compounds 7a-7f  exhibited significant antioxidant ability. The type of substituted at hydroxybenzylidene play an important role to enhance the antioxidant ability. Compound 7e exhibited higher antioxidant ability (slightly less than ascorbic acid) in both assays (DPPH and FRAP) as shown in Figure 1and  Figure 2.

Figure 1: DPPH inhibitions  % of 7a-7f   Click here to View figure

 

Compound 7d showed free radical scavenging ability more than compound 7f and that could attributed to the t-Bu group at position para reduce the antioxidant ability³⁸, the compound 7b and 7c exhibited nearly same antioxidant ability and that in agreement with most literatures. Finally compound 7a without  any substituted group around the hydroxyl group of phenol show the less antioxidant ability.

Figure 2: FRAP value of 7a-7f Click here to View figure

 

Conclusions  

A series of newly Schiff base compounds from 4-amino-1,3,4-trizole incorporating hindered phenol moieties were successfully synthesized and characterized. All of the newly compounds were tested for antioxidant activity by DPPH and FRAP assays. The antioxidant ability of these compound increases with increasing the hindered phenol.

Conflicts of Interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper

Acknowledgements    

The author would like to thank the University of Malaya for running the NMR and to NUS, (Singapore) for running the EIMS and HREIMS We are also grateful to Dr. Raied Mustafa Shakir. for his contributions and  Dr Mohammed Farouq Halabi, for his great help with screening,. Also, we would like to thank the university of Baghdad for supporting this study and provide the grant for this study.

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