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

4-amino-3-(4-(((4-hydroxy-3,5dimethoxybenzyl)oxy)methyl)phenyl)-1,2,4-triazole-5-thione was synthesized by to method the first one from melt reaction of 4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)benzoic acid with Thiocarbonyldihydrazide, the second method from convert the corresponded acid hydrazide to potassium 2-(4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl) benzoyl)hydrazinecarbodithioate salt then react with hydrazine hydrate. Newly Schiff base (7a-7f) were synthesized from reaction the 4-amino-1,2,4-triazol with substituted hydroxybenzaldehyde. The resulting compounds were characterized by IR, 1H-NMR, 13C-NMR, and HRMS data. 2,2Diphenyl-1-picrylhydrazide (DPPH) and ferric reducing antioxidant power (FRAP) assays were used to screened the antioxidant properties of the synthesized compounds. Compounds 7d , 7e and 7f exhibited significant free-radical scavenging ability in both assays. key words: 2,6-dimethoxyphenol ,4-amino-1,2,4-triazole, Schiff base, Antioxidant, DPPH, FRAP.


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 inflammatory 2 , cancers 3 , degenerative 4 and chronic diseases 5 .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 inflammatory 6 , anticancer 7 and gastroprotective 8 .
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 ð system like carotenoids 12 .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.

Chemistry
The IR spectra were obtained with a Perkin Elmer 400 Fourier Transform Infrared (FTIR) Spectrometer. 1 H and 13 C-NMR spectra were recorded at Joel Lambda spectrometers at 400 MHz) UM, Malaysia).DMSO-d 6 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.

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 ìL of 100 ìM DPPH reagent in ethanol (96%) and mixed in a 96well 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 ìL 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 equation 36

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 The 4-amino-3-(4-(((4-hydroxy-3,5dimeth -oxybenzyl)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 ÚC 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 A l l s y n t h e s i ze d c o m p o u n d s w e r e characterized by IR, 1 H-NMR, 13 C-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 Ca"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 NH 2 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 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 .asdepicted in Table 1.

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 effect 37 .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.
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 ability 38 , 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.

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.

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.